From legatekBLAH from hotmail.com Fri May 1 01:13:26 2009 From: legatekBLAH from hotmail.com (Kyle Legate) Date: Fri May 1 12:10:57 2009 Subject: EDTA for lysozyme action In-Reply-To: References: <2021705c0904251151j13b0dd5axc1a946da0f405760@mail.gmail.com> Message-ID: <75vi87F1aes3mU1@mid.individual.net> Nik_ Duke1 wrote: > > Lysozyme activity is mildly reduced in the presence of chelator, but because it is a small molecule it can do away with upto 1mM EDTA. I know lysozyme is acting only on gram positive bacteria but the practice has been carried on Gram negative bacteria also. You may want to omit lysozyme step altogether if working with E.coli. > No, lysozyme works on gram negative bacteria also, but EDTA helps lysozyme cross the LPS layer, as DK has mentioned. Brief sonication can also help lysozyme gain access to the cell wall, but there is nothing special about the cell wall that make E. coli insensitive to lysozyme. In contrast, S. aureus contains an O-acetylation of N-acetylmuramic acid that blocks the binding of lysozyme. I have to admit, I'm confused about what you mean in the second half of the first sentence I quoted above. From legatekBLAH from hotmail.com Fri May 1 01:20:34 2009 From: legatekBLAH from hotmail.com (Kyle Legate) Date: Fri May 1 12:11:02 2009 Subject: Genomic DNA isolation from mammalian cells (clarification) In-Reply-To: References: Message-ID: <75viliF1adep5U1@mid.individual.net> Dwayne Taliaferro wrote: > I am looking for a protocol that will allow me to purify genomic DNA from > mammalian cells with the purpose of using it for Southern blotting and PCR. > > DT > You can treat your cell pellet the same as a mouse tail for genomic DNA prep: http://groups.google.com/group/bionet.molbio.methds-reagnts/msg/bc55eccf64fa890c From cjtcdy from gmail.com Fri May 1 21:58:29 2009 From: cjtcdy from gmail.com (chiranjit chowdhury) Date: Sat May 2 11:37:05 2009 Subject: unknown protein's function Message-ID: <2021705c0905011958h26b2c612h5791c33968f40f22@mail.gmail.com> I am working with bacterial protein which is expressed at stationary phase. It has high level of homology with another protein. Surprisingly my protein does not show same function with that of homologue. Even my protein does not exert same physiological role that homologous protein does. The deletion of this protein does not cause any effect in bacterial growth. The data base search did not show any fruitful direction towards its function. Motif search also depicts the presence of same motifs as homologous protein contains. I made homology model of this protein and still I did not go for crystallization due to absence of proper lab set up. Can any body tell how do I find out its actual biochemical or physiological significance of that protein? The protein is present in periplasm and C-terminal is attached with cytoplasmic membrane. Is it possible to predict the probable residues which can interact with substrate or probable substrate of that protein from homology model? Please suggest any biochemical or molecular biological or any bio-informatics tools so that I can get at least any clue for its actual function Regards -- Chiranjit Chowdhury Department of Biotechnology Indian Institute of Technology, Kharagpur Kharagpur, West Bengal, India Pin: 721302 Official email: chiranjit.chowdhury@iitkgp.ac.in From shifalich from rediffmail.com Sat May 2 05:52:26 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Sat May 2 11:37:47 2009 Subject: Expression and -N end rule Message-ID: <1241007949.S.6154.37151.f4mail-234-208.rediffmail.com.1241261546.59031@webmail.rediffmail.com> Yeah! I also tgought so, just wanted second opinion out of text books. You mean to say If Bulkier groups are present next to Met, MAP will not act and Met will stay with the protein sequence? I checked the expression with coomassie and silver stain. i cudn't do western because the protein was untagged. Also, I checked in medium (Unconc. n conc.), cell lysates ( sup. n pellet both), I could not find band coressponding to my protein or any overexpressed band on the gel.I analysed abt. 6 recombinant colonies and 2 control colonies as negative ctrl.But nothing. Coomassie was blank in medium, no bands at all. Thanks Regards Shifali On Wed, 29 Apr 2009 17:55:49 +0530 wrote >Shifali, > >the N-end rule works at the cytoplasm; it has no influence on the >half-life of proteins secreted into the ER (as should be the case for >your venom protein in Pichia). Also (at least in E. coli) remember that >the amino-terminal methionine is not always removed by MAP; this depends >on the bulkiness of the side chain for the second a.a. Unsurprisingly, >residues which are destabilizing according to the N-end rule often >inhibit removal of the N-terminal Met by MAP when they are in the second >position. > >how did you check for expression in yeast? did you also test cell >lysates? Straight SDS-PAGE or Western blotting? > >> -----Original Message----- >> From: methods-bounces@oat.bio.indiana.edu >> [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of >> shifali chatrath >> Sent: Tuesday, April 21, 2009 10:46 PM >> To: methods@magpie.bio.indiana.edu >> Subject: Expression and -N end rule >> >> Dear all! >> I had mentioned my problem earlier also in the forum but >> nobody replied. I am sure you great minds should have a >> solution to my problem. Please help me if you can! >> >> I want to express a snake venom protein,~ 10kDa, 10 cys >> residues and 5 disulphide bonds.Mature protein has Arg as >> first residue after signal sequnece is clipped off. >> First of all, i tried yeast expression system. >> I expressed my protein in pPICZ alpha, under alpha factor >> signal sequence followed by Kex2 and Ste13 signal sequences, >> such that the mature protein will have native -N terminus >> with Arg as first residue, as of my protein. BUt, I could not >> see any protein expression even under different medium and >> proper aeration and whatever possible troubleshooting one could do. >> Recently, i read -N end rule which states that Arg is >> destabilizing residues and directs the protein for >> degradation to 26S proteasome. >> I checked with the company, they say, It hold true for every >> expression system. >> But, my question is, if that is the case, how the protein is >> stable in snake's venom. I have found the protein in the >> crude venom of the snake. >> Also, the protein is expressed under alpha factor signal >> sequence which is supposed to be secreted into the medium. >> Therefore,once the protein is out into medium, it is out of >> cell now, Kex2 and Ste13 cleavages should be happening >> outside the cell, therefore, there should be no proteasomal >> degradation. Am I right? >> >> After clipping off signal sequence, protein will be exported >> out, still will there be any proteasomal targetting of >> proteins with destabilizing residue at -N terminus? >> >> I read in -N end rule that, protein's life depends upon >> penultimate residue to Met. We know, for a protein to be >> synthesised, we need to have ?a start codon,i.e. Met. In -N >> end rule they say, Met will be removed be >> Metaminopeptidase(MAP), so if we express a protein we should >> not be counting Met residue in the expected MW? Because, not >> all mature protein sequences start from Met. >> >> >> Then,I tried expressing my protein using E. coli epression >> system, cDNA cloned in pET19b in Rosetta gami cells and RIL >> cells.I couldn't see any expression. Cells were growing >> normaly.(I hope, the protein is not inhibiting transcription >> and translation). Met and Gly had to be added at -N terminus >> to maintain the reading frame. >> Protein is 10Kda and was cloned without tag sequence. I mean >> expected protein should be 10kDa.I confirmed the sequences >> before cloning and even plasmid isolated from ex-pression >> hosts. DNA is there but no protein. >> >> Please answer any, if not all, of the above question. i am >> struggling with all this for alst 8 months. Your suggestions >> and help will be highly appreciable. >> >> Thanks in advance. >> >> Shifali >> >> >> >> Shifali Chatrath >> Graduate Student >> Protein science Lab >> Dept. of Biological sciences >> National University of Singapore >> Singapore >> +65-65161210 >> +65-96393449 >> _______________________________________________ >> Methods mailing list >> Methods@net.bio.net >> http://www.bio.net/biomail/listinfo/methods >> > Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From shifalich from rediffmail.com Sun May 3 22:47:07 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Mon May 4 11:00:00 2009 Subject: Western trasfer and blotting troubleshooting Message-ID: <1241007949.S.6154.37151.f4mail-234-208.rediffmail.com.replied.old.1241408827.6996@webmail.rediffmail.com> Dear All! I transfered the protein on 12% Tris -Tricine gels to Methanol wetted PVDF membrane using semi-dry apparatus at 300A, 9V for 40 min. Transfer buffer: 48mM Tris,Glycine 39mM pH(8.9) without adjusting,+ 20% methanol. After transfer, markers on one side(first lane) got beautifully tranfered but the other side of the gel (last lane),got diffused, can anybody suggest why?Anyhow, I proceeded for blotting. I dot blotted few samples on the membrane and allowed to air dry, because, a day before, these dot blots worked for these samples.I rewetted the membrane with methanol and washed with water and blocked. After Blocking in non-fat milk, I used 1:2000 anti-His mouse monoclonal, followed by 3X washes in 20mMPB +254mM Saline (PBS)+ 0.1% Tween 20 and 3X washes in PBS. Then I added 2 Ab,anti-mouse preadsorbed human, 1:5000 in Blocking buffer, it got pptd. on ice and i notice only after adding on blot. I kept the blot at 37/shaking forsing 15-20 min. and ppt. got dissolved. After 1 h incubation, I again washed as above. I noticed that two proteins from prestained marker lane are missing. Then, developed using DAB, ( Tried ECL on diffused side of the gel, last 3 lanes). I could not find any signal with both the techniques. I have standardized the antibody dilution before proceeding for this. It works.Whatever, when different I mentioned, the pptn.happened and I kept at 37. ealier, i was using 0.01% Tween 20. this time, i used 0.1% Tween 20? Then what could have gone wrong? It is the 2Ab ? Is it the pptn.? Why I see diffusion during transfer? Earlier, i had used, goat anti-mouse HRP, this time it was preadsorbed. Can anybody suggest where is the room for error that failed my expt.? Thanks REgards Shifali On Wed, 29 Apr 2009 17:55:49 +0530 wrote >Shifali, > >the N-end rule works at the cytoplasm; it has no influence on the >half-life of proteins secreted into the ER (as should be the case for >your venom protein in Pichia). Also (at least in E. coli) remember that >the amino-terminal methionine is not always removed by MAP; this depends >on the bulkiness of the side chain for the second a.a. Unsurprisingly, >residues which are destabilizing according to the N-end rule often >inhibit removal of the N-terminal Met by MAP when they are in the second >position. > >how did you check for expression in yeast? did you also test cell >lysates? Straight SDS-PAGE or Western blotting? > >> -----Original Message----- >> From: methods-bounces@oat.bio.indiana.edu >> [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of >> shifali chatrath >> Sent: Tuesday, April 21, 2009 10:46 PM >> To: methods@magpie.bio.indiana.edu >> Subject: Expression and -N end rule >> >> Dear all! >> I had mentioned my problem earlier also in the forum but >> nobody replied. I am sure you great minds should have a >> solution to my problem. Please help me if you can! >> >> I want to express a snake venom protein,~ 10kDa, 10 cys >> residues and 5 disulphide bonds.Mature protein has Arg as >> first residue after signal sequnece is clipped off. >> First of all, i tried yeast expression system. >> I expressed my protein in pPICZ alpha, under alpha factor >> signal sequence followed by Kex2 and Ste13 signal sequences, >> such that the mature protein will have native -N terminus >> with Arg as first residue, as of my protein. BUt, I could not >> see any protein expression even under different medium and >> proper aeration and whatever possible troubleshooting one could do. >> Recently, i read -N end rule which states that Arg is >> destabilizing residues and directs the protein for >> degradation to 26S proteasome. >> I checked with the company, they say, It hold true for every >> expression system. >> But, my question is, if that is the case, how the protein is >> stable in snake's venom. I have found the protein in the >> crude venom of the snake. >> Also, the protein is expressed under alpha factor signal >> sequence which is supposed to be secreted into the medium. >> Therefore,once the protein is out into medium, it is out of >> cell now, Kex2 and Ste13 cleavages should be happening >> outside the cell, therefore, there should be no proteasomal >> degradation. Am I right? >> >> After clipping off signal sequence, protein will be exported >> out, still will there be any proteasomal targetting of >> proteins with destabilizing residue at -N terminus? >> >> I read in -N end rule that, protein's life depends upon >> penultimate residue to Met. We know, for a protein to be >> synthesised, we need to have ?a start codon,i.e. Met. In -N >> end rule they say, Met will be removed be >> Metaminopeptidase(MAP), so if we express a protein we should >> not be counting Met residue in the expected MW? Because, not >> all mature protein sequences start from Met. >> >> >> Then,I tried expressing my protein using E. coli epression >> system, cDNA cloned in pET19b in Rosetta gami cells and RIL >> cells.I couldn't see any expression. Cells were growing >> normaly.(I hope, the protein is not inhibiting transcription >> and translation). Met and Gly had to be added at -N terminus >> to maintain the reading frame. >> Protein is 10Kda and was cloned without tag sequence. I mean >> expected protein should be 10kDa.I confirmed the sequences >> before cloning and even plasmid isolated from ex-pression >> hosts. DNA is there but no protein. >> >> Please answer any, if not all, of the above question. i am >> struggling with all this for alst 8 months. Your suggestions >> and help will be highly appreciable. >> >> Thanks in advance. >> >> Shifali >> >> >> >> Shifali Chatrath >> Graduate Student >> Protein science Lab >> Dept. of Biological sciences >> National University of Singapore >> Singapore >> +65-65161210 >> +65-96393449 >> _______________________________________________ >> Methods mailing list >> Methods@net.bio.net >> http://www.bio.net/biomail/listinfo/methods >> > Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From engelbert_buxbaum from hotmail.com Mon May 4 13:24:10 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon May 4 14:21:01 2009 Subject: Western trasfer and blotting troubleshooting References: Message-ID: Am 03.05.2009, 23:47 Uhr, schrieb shifali chatrath : > I transfered the protein on 12% Tris -Tricine gels to Methanol wetted > PVDF membrane using semi-dry apparatus at 300A, 9V for 40 min. > Transfer buffer: 48mM Tris,Glycine 39mM pH(8.9) without adjusting,+ 20% > methanol. > After transfer, markers on one side(first lane) got beautifully > tranfered but the other side of the gel (last lane),got diffused, can > anybody suggest why?Anyhow, I proceeded for blotting. There is little point in proceeding with ab staining, if the transfer did not work. After wetting the PVDF with MeOH you need to transfer to blotting buffer, as MeOH is not conductive. The blotting sandwich needts to be assembled without air bubbles. Are the electrodes of the semi-dry blotter ok (not unevenly worn)? > 3X washes in PBS. Then I added 2 Ab,anti-mouse preadsorbed human, 1:5000 > in Blocking buffer, it got pptd. on ice and i notice only after adding > on blot. Well, it shouldn't, so you must have done something wrong. The precipitate is probably not the antibody, as the concentration is too low to cause a visible precipitate. Redo the blocking buffer, making sure that you get the right chemicals in the correct amounts. If that doesn't get rid of the precipitation, leave the compounds out one by one to find the culprit. It could be some contaminant, or, in very rare cases, a bad batch of chemicals (Sigma once send me "SDS" that was water insoluble). Alternative option: consider publishing in the "Journal of Irreproducible Science" ;-) From shifalich from rediffmail.com Tue May 5 03:28:59 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Tue May 5 08:55:55 2009 Subject: Western trasfer and blotting troubleshooting Message-ID: <1241465080.S.3859.8399.f4mail-234-222.rediffmail.com.old.1241512139.65177@webmail.rediffmail.com> On Tue, 05 May 2009 00:54:40 +0530 wrote >Am 03.05.2009, 23:47 Uhr, schrieb shifali ?chatrath ? >: > > >> I transfered the protein on 12% Tris -Tricine gels to Methanol wetted ? >> PVDF membrane using semi-dry apparatus at 300A, 9V for 40 min. >> Transfer buffer: 48mM Tris,Glycine 39mM pH(8.9) without adjusting,+ 20% ? >> methanol. >> After transfer, markers on one side(first lane) got beautifully ? >> tranfered but the other side of the gel (last lane),got diffused, can ? >> anybody suggest why?Anyhow, I proceeded for blotting. > >There is little point in proceeding with ab staining, if the transfer did ? >not work. After wetting the PVDF with MeOH you need to transfer to ? >blotting buffer, as MeOH is not conductive. The blotting sandwich needts ? >to be assembled without air bubbles. Are the electrodes of the semi-dry ? >blotter ok (not unevenly worn)? How to check if the electrodes are OK? I did soak the membrane in transfer buffer before transfer. > >> 3X washes in PBS. Then I added 2 Ab,anti-mouse preadsorbed human, 1:5000 ? >> in Blocking buffer, it got pptd. on ice and i notice only after adding ? >> on blot. > >Well, it shouldn't, so you must have done something wrong. The precipitate ? >is probably not the antibody, as the concentration is too low to cause a ? >visible precipitate. Redo the blocking buffer, making sure that you get ? >the right chemicals in the correct amounts. If that doesn't get rid of the ? >precipitation, leave the compounds out one by one to find the culprit. It ? >could be some contaminant, or, in very rare cases, a bad batch of ? >chemicals (Sigma once send me "SDS" that was water insoluble). because of milk products in blocking buffer which can be dissolved at 37,I mentioned this because after secondary Ab incubation, 2 of the pretsained markers were missing from the membrane.So, I was wonderin if temp. is the reason for loss of proteins frm. the membrane or wht? I understand that the pptn. is not because of Ab, but > >Alternative option: consider publishing in the "Journal of Irreproducible ? Is there any such journal? I have many manuscripts to publish then... :)) >Science" ;-) >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods > Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From Burgdorfr from ukzn.ac.za Wed May 6 07:13:25 2009 From: Burgdorfr from ukzn.ac.za (Richard Burgdorf) Date: Wed May 6 08:04:50 2009 Subject: Bisbenzimide-PEG/HA-Yellow&In-Reply-To= Message-ID: <4A019B64.2AB3.0006.1@ukzn.ac.za> Hi, Does anyone know how to put this together? I can't find a supplier for the final product but can get hold of bisbenzimide and PEG 6000. Thanks Richard Please find our Email Disclaimer here: http://www.ukzn.ac.za/disclaimer/ From yvonne.couch from pharm.ox.ac.uk Wed May 6 11:35:51 2009 From: yvonne.couch from pharm.ox.ac.uk (Yvonne Couch) Date: Wed May 6 11:59:32 2009 Subject: Peptide in solution Message-ID: <003a01c9ce68$b7db1a00$27914e00$@couch@pharm.ox.ac.uk> I have a peptide dissolved in PBS at 1mg/ml, I want to concentrate it, is it possible to get the peptide out of solution in order to concentrate it? Cheers Yvonne From nick.theodorakis from gmail.com Wed May 6 13:12:58 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Wed May 6 15:22:27 2009 Subject: Peptide in solution References: Message-ID: <6269a292-fc95-4c3c-9187-dc0e787dd94d@s21g2000vbb.googlegroups.com> On May 6, 12:35?pm, "Yvonne Couch" wrote: > I have a peptide dissolved in PBS at 1mg/ml, I want to concentrate it, is it > possible to get the peptide out of solution in order to concentrate it? > > Cheers > > Yvonne You could bind it to a (for example) C-18 column, elute it with a volatile eluant such as acetonitrile, and lyophilize it. There are some disposable C-18 cartridges called Sep-Pak made by Waters that might be suitable. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From bmacgreg from unc.edu Wed May 6 14:57:01 2009 From: bmacgreg from unc.edu (Barbara MacGregor) Date: Wed May 6 15:22:33 2009 Subject: The elusive HA Yellow In-Reply-To: <200905061704.n46H4Wb18096@net.bio.net> References: <200905061704.n46H4Wb18096@net.bio.net> Message-ID: <0DA2C10F-C0C8-4A0A-9CB2-7A30C1460CEC@unc.edu> Hello, Actually I eventually found a supplier for HA Yellow (and Red!), at least they had it a couple of years ago. Try the Resolve-It kit from Vector Laboratories (Burlingame CA). Barbara > > > From: "Richard Burgdorf" > Date: May 6, 2009 8:13:25 AM EDT > To: > Subject: Bisbenzimide-PEG/HA-Yellow&In-Reply-To= > > > Hi, > > Does anyone know how to put this together? I can't find a supplier > for the final product but can get hold of bisbenzimide and PEG 6000. > > Thanks > Richard > > Please find our Email Disclaimer here: http://www.ukzn.ac.za/disclaimer/ > > > > > From Paul.Phelan from tufts.edu Wed May 6 13:03:44 2009 From: Paul.Phelan from tufts.edu (Paul J. Phelan) Date: Wed May 6 15:22:41 2009 Subject: Peptide in solution In-Reply-To: <003a01c9ce68$b7db1a00$27914e00$%couch@pharm.ox.ac.uk> References: <003a01c9ce68$b7db1a00$27914e00$%couch@pharm.ox.ac.uk> Message-ID: <20090506140344.aa7idsyps0g884wk@webmail.tufts.edu> I would concentrate the peptide in a "Speed-vac": you freeze it and then put it in a centrifuge under vacuum, the solvent will evaporate, but the PBS salts will be concentrated along with the peptide. Otherwise, you could lyophilize the peptide and re-dissolve it with H2O, to whatever volume you like. Quoting Yvonne Couch : > I have a peptide dissolved in PBS at 1mg/ml, I want to concentrate it, is it > possible to get the peptide out of solution in order to concentrate it? > > Cheers > > Yvonne > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From novalidaddress from nurfuerspam.de Wed May 6 15:28:47 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Wed May 6 18:45:28 2009 Subject: Peptide in solution References: <6269a292-fc95-4c3c-9187-dc0e787dd94d@s21g2000vbb.googlegroups.com> Message-ID: Hi Nick, will your suggestion also work for larger peptides i.e. proteins? Will MeCN still work as eluent? Wo > > You could bind it to a (for example) C-18 column, elute it with a > volatile eluant such as acetonitrile, and lyophilize it. There are > some disposable C-18 cartridges called Sep-Pak made by Waters that > might be suitable. > > Nick > > -- > Nick Theodorakis > nick_theodora...@hotmail.com > contact form:http://theodorakis.net/contact.html From oliver.starks from gmail.com Thu May 7 13:32:20 2009 From: oliver.starks from gmail.com (oliver.starks@gmail.com) Date: Fri May 8 08:55:37 2009 Subject: Coomassie safety Message-ID: Some Bradford assay cuvettes were improperly dumped into a bacterial broth waste bottle in our lab, and subsequently autoclaved. How much of a concern is the especially unpleasant odor that was created? Is Coomassie dangerous if heated in this manner? From yvonne.couch from pharm.ox.ac.uk Fri May 8 04:28:41 2009 From: yvonne.couch from pharm.ox.ac.uk (Yvonne Couch) Date: Fri May 8 08:55:50 2009 Subject: Peptide in solution In-Reply-To: <20090506140344.aa7idsyps0g884wk@webmail.tufts.edu> References: <003a01c9ce68$b7db1a00$27914e00$%couch@pharm.ox.ac.uk> <20090506140344.aa7idsyps0g884wk@webmail.tufts.edu> Message-ID: <003e01c9cfbf$601127d0$20337770$@couch@pharm.ox.ac.uk> Hi all, Thanks for the brilliant advice and this seems silly now but I don't know how I would go about lyophilizing a peptide, can anyone help? Cheers again! Yvonne -----Original Message----- From: Paul J. Phelan [mailto:Paul.Phelan@tufts.edu] Sent: 06 May 2009 19:04 To: Yvonne Couch Cc: methods@magpie.bio.indiana.edu Subject: Re: Peptide in solution I would concentrate the peptide in a "Speed-vac": you freeze it and then put it in a centrifuge under vacuum, the solvent will evaporate, but the PBS salts will be concentrated along with the peptide. Otherwise, you could lyophilize the peptide and re-dissolve it with H2O, to whatever volume you like. Quoting Yvonne Couch : > I have a peptide dissolved in PBS at 1mg/ml, I want to concentrate it, is it > possible to get the peptide out of solution in order to concentrate it? > > Cheers > > Yvonne > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From Sharon.Waldrop from utsouthwestern.edu Fri May 8 09:22:23 2009 From: Sharon.Waldrop from utsouthwestern.edu (Sharon Waldrop) Date: Fri May 8 10:04:23 2009 Subject: Another source for Invitrogen YO-PRO-1 Message-ID: <4A03F9CF0200003E000483A4@swnw126.swmed.org> Hello everyone, Hope you can help. Invitrogen has a product they call YO-PRO-1 (monomeric cyanine nucleic acid stain / carbocyanine nucleic acid stain - green fluorescent (491/509)). Is there another source for this dye? I have tried Googling and talking to other companies, no luck. I am going nuts having to deal with Invitrogen. Thanks for your help, Shar Waldrop Univ. of TX Southwestern Medical Center Dallas, TX 75390 From nick.theodorakis from gmail.com Fri May 8 11:12:47 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Fri May 8 14:21:58 2009 Subject: Peptide in solution References: <6269a292-fc95-4c3c-9187-dc0e787dd94d@s21g2000vbb.googlegroups.com> Message-ID: <76332ebd-cab7-486e-84ae-d2b37ad8e47f@r13g2000vbr.googlegroups.com> On May 6, 4:28?pm, WS wrote: > Hi Nick, > > will your suggestion also work for larger peptides i.e. proteins? Will > MeCN still work as eluent? > Wo > The best answer I can give is "maybe" or even "probably." If enzymatic or other biological activity is important to you, it may not be the best way, though. For a protein, the first thing I would try is to concentrate by ultrafiltration. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From nick.theodorakis from gmail.com Fri May 8 11:19:41 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Fri May 8 14:22:13 2009 Subject: Peptide in solution References: <003a01c9ce68$b7db1a00$27914e00$%couch@pharm.ox.ac.uk> <20090506140344.aa7idsyps0g884wk@webmail.tufts.edu> Message-ID: <0833fc26-dc3e-426d-8fcc-4b4b7cd96d61@q14g2000vbn.googlegroups.com> On May 8, 5:28?am, "Yvonne Couch" wrote: > Hi all, > Thanks for the brilliant advice and this seems silly now but I don't know > how I would go about lyophilizing a peptide, can anyone help? > Cheers again! > Yvonne > A lyophilizer is simply a freeze-dryer, which is a vacuum pump and cold trap hooked up to a chamber in which the samples are placed. A Speed-Vac is a lyophilizer in which the chamber contains a centrifuge and is more suitable for small sample volumes. To lyophilze, just freeze the sample at low temperature (dry ice bath or -80C freezer), put it in the chamber, and apply the vaccum. If you are borrowing someone's make sure whatver solvent you are using is ok with them; some people are fussy about what goes in their cold trap. You might have better luck if yo need to borrow to find someone who is doing HPLC since they are probably already drying down samples that way. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From engelbert_buxbaum from hotmail.com Fri May 8 14:19:13 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri May 8 15:26:17 2009 Subject: Western trasfer and blotting troubleshooting References: Message-ID: Am 05.05.2009, 04:28 Uhr, schrieb shifali chatrath : >> Are the electrodes of the semi-dry blotter ok (not unevenly worn)? > > How to check if the electrodes are OK? eyeball it, if it looks ok, that's sufficient. >> Alternative option: consider publishing in the "Journal of >> Irreproducible Science" ;-) > > Is there any such journal? I have many manuscripts to publish then... :)) who hasn't? From engelbert_buxbaum from hotmail.com Fri May 8 14:25:52 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri May 8 15:26:22 2009 Subject: Bisbenzimide-PEG/HA-Yellow&In-Reply-To= References: Message-ID: Am 06.05.2009, 08:13 Uhr, schrieb Richard Burgdorf : > Does anyone know how to put this together? I can't find a supplier for > the final product but can get hold of bisbenzimide and PEG 6000. PEG is polyethylene glycol and can be found as such in the Fluka catalogue. 6000 is the average molecular weight. Bisbenzimide is an intercalating fluorescent stain that is also in the Fluka catalogue. Be careful with that stuff, you don't want it to intercalate into your own DNA. Have somebody train you before working with such material. From engelbert_buxbaum from hotmail.com Fri May 8 14:31:07 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri May 8 15:26:26 2009 Subject: Peptide in solution References: Message-ID: Am 06.05.2009, 12:35 Uhr, schrieb Yvonne Couch : > I have a peptide dissolved in PBS at 1mg/ml, I want to concentrate it, > is it > possible to get the peptide out of solution in order to concentrate it? Put the solution into a dialysis bag and put some PEG 20,000 outside. The stuff is hygroscopic and sucks the water out of the bag. Dry Sephadex works to, but is more expensive. Just look after the thing from time to time, you don't want all the water removed from your sample. Centrifugal concentrators (e.g. Millipore) or Amicon pressure cells also work. From engelbert_buxbaum from hotmail.com Fri May 8 14:35:29 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri May 8 15:26:31 2009 Subject: Coomassie safety References: Message-ID: Am 07.05.2009, 14:32 Uhr, schrieb : > Some Bradford assay cuvettes were improperly dumped into a bacterial > broth waste bottle in our lab, and subsequently autoclaved. How much > of a concern is the especially unpleasant odor that was created? Is > Coomassie dangerous if heated in this manner? No. Were those polystyrene cuvettes? In that case the smell probably comes from decomposing plastic. I would not make a habit of breathing styrene on a regular basis, but a minor amount in a well ventilated area (!?) would not make you keel over. From engelbert_buxbaum from hotmail.com Fri May 8 14:47:19 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri May 8 15:26:46 2009 Subject: Another source for Invitrogen YO-PRO-1 References: Message-ID: Am 08.05.2009, 10:22 Uhr, schrieb Sharon Waldrop : > Hope you can help. Invitrogen has a product they call YO-PRO-1 > (monomeric cyanine nucleic acid stain / carbocyanine nucleic acid stain > - green fluorescent (491/509)). Is there another source for this dye? I > have tried Googling and talking to other companies, no luck. I am going > nuts having to deal with Invitrogen. That bad, eh? Yo-Pro-1 is a trade name, first you have to find out exactly what compound it is (catalogue, patent applications, literature databases etc). Helpful is the CAS (Chemical Abstracts) number, if you can locate that. Then you can look which other manufacturer sells the same stuff, under a different trade name of course. Often it helps to simply Google for the CAS-number. Or you simply say "What the heck" and use a different stain. There are plenty of those out there, and for some the composition is a matter of public record. That is my preferred solution, as non-disclosed components IMHO violate the requirement that science should be reproducible. Therefore I never use them, on principle. From huang_wen from ustc.edu Fri May 8 15:09:59 2009 From: huang_wen from ustc.edu (Wen Huang) Date: Fri May 8 15:26:53 2009 Subject: Primer extension kinetics In-Reply-To: <9dbf8a79-693b-4b56-b7dd-9d3ced4451bf@j38g2000yqa.googlegroups.com> References: <9dbf8a79-693b-4b56-b7dd-9d3ced4451bf@j38g2000yqa.googlegroups.com> Message-ID: Hi Does anybody know the kinetics of primer extension reaction? Is it linear? A related question is, under the temperature of a primer extension reaction, does the primer-template hybrid gets disassociated? If they do, to what extent, in other words, what magnitude is K as compared to k in the primer extension step. Thanks a lot. Wen From m.fuchs from qub.ac.uk Mon May 11 10:51:24 2009 From: m.fuchs from qub.ac.uk (Marc-Aurel Fuchs) Date: Mon May 11 12:45:16 2009 Subject: smart oligo Message-ID: <1BD31E7AA79D7E42BB1B654E1F9A695A04CBB65C9D@EX2K7-VIRT-1.ads.qub.ac.uk> Can you replace the SMART oligo that comes with the Clontech RACE kit with a normal DNA primer of the same sequence? Or does it have to be an RNA oligo? Thanks, fuchse From martino from metabolix.com Mon May 11 14:50:15 2009 From: martino from metabolix.com (Matthew Martino) Date: Mon May 11 18:12:15 2009 Subject: problems with restriction digestion Message-ID: <03BF7D1E64258941A344815B1C1184A942EF3160@mbx-exchmb01> I agree. There is always the concern of Methylation, a cells natural defense of dna from restriction enzymes. If the extra bands appear and they seem faint, for instance dimmer than the band above and the band below it, then you are having partials. This can easily be explained by the fact that some enzymes require exact salt conditions for activity and anything differing from the optimum will result in partial digests. One way to check if this is the case is doing digests with the enzymes individually. You should see linear DNA fragments in both cases (slight smiling of bands, but no streaking and definitely not multiple bands such as linear and supercoiled running together). Whichever enzymes doesn't give you completely linear DNA is the problem. Matt From pjie2 from cam.ac.uk Mon May 11 17:44:35 2009 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Mon May 11 18:12:25 2009 Subject: smart oligo In-Reply-To: References: Message-ID: <76ro2qF1epc8eU1@mid.individual.net> DK wrote: > In article , Marc-Aurel Fuchs wrote: >> Can you replace the SMART oligo that comes with the Clontech RACE kit with a >> normal DNA primer of the same sequence? > > No reason why you can't. Just to be on a safer side, may consider > using gel/HPLC purified oligo. I haven't tried it personally, but my understanding is that this is incorrect. The SMART template-switching primer is a hybrid oligo - the majority is DNA, but the last three bases are RNA. So far as I know, the template-switching only works with a hybrid oligo, not with a pure RNA or a pure DNA oligo. Peter From kuforen from gmail.com Mon May 11 20:02:29 2009 From: kuforen from gmail.com (Seong Hwan Park) Date: Tue May 12 08:31:55 2009 Subject: smart oligo In-Reply-To: <1BD31E7AA79D7E42BB1B654E1F9A695A04CBB65C9D@EX2K7-VIRT-1.ads.qub.ac.uk> References: <1BD31E7AA79D7E42BB1B654E1F9A695A04CBB65C9D@EX2K7-VIRT-1.ads.qub.ac.uk> Message-ID: <7e2b93f90905111802p113a9dd7wfe3dbb83eab869d3@mail.gmail.com> The SMART oligo has three RNA bases at 3' end. However, I've read an article about cDNA library technique, which describes that authors used just DNA bases at 3' end of SMART oligo and got a good result. I think d(GGG) instead of r(GGG) will also work, but less efficiently. In the patent document of SMART oligo, addition of phosphate to the 3' end of r(GGG) made the background clean. Seong 2009/5/12 Marc-Aurel Fuchs : > Can you replace the SMART oligo that comes with the Clontech RACE kit with a normal DNA primer of the same sequence? > Or does it have to be an RNA oligo? > > Thanks, ?fuchse > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Seong Hwan Park, M.D., Ph.D. Instructor in Legal Medicine, College of Medicine, Korea University 126-1 Anadong 5ga, Seongbukgu, Seoul 136-704 South Korea E-mail: kuforen@gmail.com or pelvis@korea.ac.kr Tel: 82-2-920-6158 Fax: 82-2-928-3901 From shifalich from rediffmail.com Tue May 12 04:32:14 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Tue May 12 08:32:04 2009 Subject: proteomics problem Message-ID: <20090512093214.58410.qmail@f4mail-235-131.rediffmail.com> Dear all! My colleague has the following query. If anybody knows about it, please reply. I am sequencing a protein of mass 6771Da by N-terminal sequencing using pulse liquid method. I dissolved my freeze dried protein in 25% acetonitrile and spotted on to the glass fibre filter that was previously spotted with polybrene.I then dried the catridge under the flow of argon. Surprisingly i could see the development of red spots on the glass fibre filter. Can any one give me a probable explanation for this observation and tell me whether this will affect my sequencing? An important thing to note is that this red color development was observed for this protein only among the others that i have sequenced previously. Thanks Regards Shifali Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From SBrown from ccia.unsw.edu.au Tue May 12 06:35:06 2009 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Tue May 12 08:32:45 2009 Subject: MMTV as a reponsive element is a lucifierase reporter construct Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A07D3B628@mail01.ccia.local> Hi, I am aware of MMTV being a responsive element to glucocorticoids and androgens when in a lucifierase reporter construct. Does anyone know if MMTV is responsive to progesterone and oestrogen? Regards Scott Scott Brown PhD Candidate Molecular Carcinogenesis Program Children's Cancer Institute of Australia for Medical Research High Street (PO Box 81) RANDWICK NSW 2031 AUSTRALIA Phone: +61 2 9382 1829 Fax: +61 2 9382 1850 Email: sbrown@ccia.unsw.edu.au Web: www.ccia.org.au Children's Cancer Institute Australia is the only independent medical research institute in Australia solely devoted to research into the causes, prevention and cure of childhood cancer. Our vision is to save the lives of all children with cancer and eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error, please contact the sender immediately and destroy the original message. Children's Cancer Institute Australia for Medical Research is the only independent medical research institute in Australia devoted to research into the causes, prevention, better treatment and ultimately a cure of childhood cancer. Our vision is to save the lives of all children with cancer and to eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error please contact the sender immediately and destroy the original message. From SBrown from ccia.unsw.edu.au Tue May 12 09:29:29 2009 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Tue May 12 13:23:27 2009 Subject: Real time pcr multiplex with probe vs. sybrgreen Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A07D3B62D@mail01.ccia.local> Hi Does anyone know the pros and cons of multiplexing and using probes for real time as opposed to using sybrgreen master mix? S Scott Brown PhD Candidate Molecular Carcinogenesis Program Children's Cancer Institute of Australia for Medical Research High Street (PO Box 81) RANDWICK NSW 2031 AUSTRALIA Phone: +61 2 9382 1829 Fax: +61 2 9382 1850 Email: sbrown@ccia.unsw.edu.au Web: www.ccia.org.au Children's Cancer Institute Australia is the only independent medical research institute in Australia solely devoted to research into the causes, prevention and cure of childhood cancer. Our vision is to save the lives of all children with cancer and eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error, please contact the sender immediately and destroy the original message. Children's Cancer Institute Australia for Medical Research is the only independent medical research institute in Australia devoted to research into the causes, prevention, better treatment and ultimately a cure of childhood cancer. Our vision is to save the lives of all children with cancer and to eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error please contact the sender immediately and destroy the original message. From aawara from pontiff-playground.org Tue May 12 20:53:08 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Wed May 13 09:25:51 2009 Subject: MMTV as a reponsive element is a lucifierase reporter construct References: Message-ID: <8KpOl.19663$hX2.7774@newsfe19.iad> In , Scott Brown wrote: > I am aware of MMTV being a responsive element to > glucocorticoids and androgens when in a lucifierase reporter > construct. Does anyone know if MMTV is responsive to > progesterone and oestrogen? The MMTV LTR is responsive to glucocorticoids, some androgens (eg. dihydrotestosterone), and progesterone. It is not responsive to estrogens. This lack of responsiveness is very significant for the biology of mouse mammary tumor virus (MMTV). I would check papers by Stan McKnight from the mid to late 80's. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From jenkinsr from umich.edu Tue May 12 22:46:01 2009 From: jenkinsr from umich.edu (Ron Jenkins) Date: Wed May 13 09:26:01 2009 Subject: pET23d vector Message-ID: <4A0A4279.6050102@umich.edu> Aki, Have you tried expressing it in TB media, and/or BL21 AI cells. Arabinose inducible tend to be less leaky which seems to help for insoluble proteins. Further, TB media does wonders. If this doesn't work check your pKa and check to the pH of your media. Buffered media (such as TB) which is richer tend to work better. Ron From blackhole from abuse.plus.com Wed May 13 10:35:19 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Wed May 13 11:33:43 2009 Subject: Real time pcr multiplex with probe vs. sybrgreen References: Message-ID: <7I4Dr4C3iuCKFAUh@abuse.plus.com> Historians believe that in newspost on Wed, 13 May 2009, Scott Brown penned the following literary masterpiece: > >Does anyone know the pros and cons of multiplexing and using probes for real time as opposed to using sybrgreen master mix? Ask on the qpcrlistserv yahoogroup Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From rafaelescateperu from gmail.com Wed May 13 12:18:11 2009 From: rafaelescateperu from gmail.com (rafael escate) Date: Wed May 13 13:57:46 2009 Subject: Hello - Thrombin Cleavage of GST Message-ID: Hello Dra Lyle Najita How are you? I would like know about the information of thrombin cleavage of GST. Could you say me some protocol to use the thrombin? and this way have my protein. for example dates as temperature and performance condicion. I will wait your request with great interest first of all thanks for your time Rafael* * From TieQiao.Wu from bakeridi.edu.au Wed May 13 21:44:12 2009 From: TieQiao.Wu from bakeridi.edu.au (TieQiao Wu) Date: Thu May 14 14:12:40 2009 Subject: Methods Digest, Vol 48, Issue 10 In-Reply-To: <200905131705.n4DH5Wb16373@net.bio.net> References: <200905131705.n4DH5Wb16373@net.bio.net> Message-ID: <217613C88C00D4448B099AD7A374F253921D74@exch01.bhri.internal> Hi Does anyone have some experiences to purify insoluble GST-fusion protein? I tried to purify GST-fusion protein recently, but it looks that the protein did not bind to the beads, I did coommassie stain and western blotting and results show the protein was expressed. I purified the same protein last year although the fusion protein degraded partially. In the cell suspension 1M DTT and 10% Sarkosyl were added before sonication, 10% Triton X-100 was added after sonication, the final concentrations are: 0.7% Sarkosyl, 2% Triton X-100 and 5mM DTT. Thanks a lot! Tieqiao This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you have received this email in error please promptly delete this email and notify the system manager. This email has been scanned by the MessageLabs Email Security System. For more information please visit http://www.messagelabs.com/email From SBrown from ccia.unsw.edu.au Thu May 14 07:09:15 2009 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Thu May 14 14:13:11 2009 Subject: molecular biology textbooks Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A07D3B63B@mail01.ccia.local> Can anybody recommend a textbook that is a good reference for techniques used in the lab, i know everything is available on the web but i'm still a bit old school and prefer to look over a book. S Scott Brown PhD Candidate Molecular Carcinogenesis Program Children's Cancer Institute of Australia for Medical Research High Street (PO Box 81) RANDWICK NSW 2031 AUSTRALIA Phone: +61 2 9382 1829 Fax: +61 2 9382 1850 Email: sbrown@ccia.unsw.edu.au Web: www.ccia.org.au Children's Cancer Institute Australia is the only independent medical research institute in Australia solely devoted to research into the causes, prevention and cure of childhood cancer. Our vision is to save the lives of all children with cancer and eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error, please contact the sender immediately and destroy the original message. Children's Cancer Institute Australia for Medical Research is the only independent medical research institute in Australia devoted to research into the causes, prevention, better treatment and ultimately a cure of childhood cancer. Our vision is to save the lives of all children with cancer and to eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error please contact the sender immediately and destroy the original message. From shifalich from rediffmail.com Thu May 14 10:50:20 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Thu May 14 14:13:16 2009 Subject: proteomics Message-ID: <1241814436.S.2802.18201.f4mail-234-125.rediffmail.com.old.1242316220.47812@webmail.rediffmail.com> Dear all! My colleague has the following query. If anybody knows about it, please reply. I am sequencing a protein of mass 6771Da by N-terminal sequencing using pulse liquid method. I dissolved my freeze dried protein in 25% acetonitrile and spotted on to the glass fibre filter that was previously spotted with polybrene.I then dried the catridge under the flow of argon. Surprisingly i could see the development of red spots on the glass fibre filter. Can any one give me a probable explanation for this observation and tell me whether this will affect my sequencing? An important thing to note is that this red color development was observed for this protein only among the others that i have sequenced previously. Thanks Regards Shifali Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From sakkthi18 from gmail.com Thu May 14 21:39:04 2009 From: sakkthi18 from gmail.com (Sakkthi subramaniyam) Date: Fri May 15 11:17:45 2009 Subject: Acid phosphatase Message-ID: Hai can any one give me complete procedure for estimation of ACID PHOSPHATASE, INVERTASE AND SUCROSE SYNTHASE with calculation formula. Please dude I got procedures but I couldn't sort out how to arrive mathematically.Here are references for those estimations ACID PHOSPHATASE, Parfsh, R.W. (1974). J. Histochem. Cytochem., 9 : 542. INVERTASE Sridhar, R. and S.H. Ou. (1972). Phillippine Phytopath., 8, 52-56. SUCROSE SYNTHASE Wardlaw, I.F. and J. Willenbrink (1994). Aust. J. Plant Physiol, 21,255-271. -- With regards, Sakkthi subramaniyam, II. M.Sc.(Ag) From stanleycheungkk from yahoo.com Fri May 15 06:17:15 2009 From: stanleycheungkk from yahoo.com (Stanley Cheung) Date: Fri May 15 11:27:14 2009 Subject: Monomeric Abeta detection in brain of APP tg mice using western blotting Message-ID: <675919.22338.qm@web54505.mail.re2.yahoo.com> Hi there, Does anyone have experiences in detecting and quantifying soluble monomeric Abeta in brain of APP transgenic mice (Tg2576). There is no clear description on the loading amount in journals. I tried many times and eventually found a paper described using 100ug to 250ug of brain homogenate in TBS after ultracenfriguation, 100k xg. However, I believe there is a limited total protein concentration which can be extracted using TBS during homogenization. Actually, 250ug sample means too large volume for loading?on mini-SDS-PAGE gel. If for detecting monomeric Abeta (~4KD), the volume of the loading sample should be very small for better resolution. I tried to use TCA precipitation to reduce the volume, but the monomeric Abeta disappeared and the?precipitate cannot be dissolved completely. Compared with samples without ultracentrifugation (using the sample TCA precipitation method) and?the pellet obtained after ultracentrifugation, monomeric Abeta can be seen in these samples on the?blot. It makes me to think that TCA precipitation causes too much protein loss for small peptide or the abundance of the peptide in the sample is too little. I should not use the whole brain for doing western blotting of Abeta, it consumes too much sample. I found most papers only use ELISA for soluble Abeta detection, or immunoprecipitate the Abeta from large amount of sample and load it on the gel for WB. However, these two methods are too expensive in my lab. Lyophilization doesn't work too, because it increases the salt content too much which distorts the bands in the gel and the lyophilized sample cannot be totally dissolved in reduced volume. Can anyone suggest the loading amount and ways to concentrate the sample for WB detection? Or is it practical to use WB for such detection? Thank you very much for your opinion in advance. Sincerely, Stanley PhD Candidate Department of Medicine and Therapeutics Faculty of Medicine Chinese University of Hong Kong Send instant messages to your online friends http://uk.messenger.yahoo.com From engelbert_buxbaum from hotmail.com Fri May 15 11:28:13 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri May 15 12:35:13 2009 Subject: Methods Digest, Vol 48, Issue 10 References: <200905131705.n4DH5Wb16373@net.bio.net> Message-ID: Am 13.05.2009, 22:44 Uhr, schrieb TieQiao Wu : > Hi > > Does anyone have some experiences to purify insoluble GST-fusion > protein? I tried to purify GST-fusion protein recently, but it looks > that the protein did not bind to the beads, I did coommassie stain and > western blotting and results show the protein was expressed. That can happen, and you have two options: - use a differnet tag (or a different position of tag, N- vs C-terminal) and hope that it will work - do a conventional purification (IEC, SEC, HIC etc) From engelbert_buxbaum from hotmail.com Fri May 15 11:33:08 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri May 15 12:35:19 2009 Subject: molecular biology textbooks References: Message-ID: Am 14.05.2009, 08:09 Uhr, schrieb Scott Brown : > > Can anybody recommend a textbook that is a good reference for techniques > used in the lab, i know everything is available on the web but i'm still > a bit old school and prefer to look over a book. That depends on what, precisely, you mean by "Molecualr Biology". If you just mean "gene fiddeling" tha Maniatis is probably still your best bet. If you mean "Biology on a molecular level", than no, there is no single book covering the techniques. Even the "Methods in Molecular Biology" series only covers part of it. From engelbert_buxbaum from hotmail.com Fri May 15 11:49:27 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri May 15 18:09:02 2009 Subject: Acid phosphatase References: Message-ID: Am 14.05.2009, 22:39 Uhr, schrieb Sakkthi subramaniyam : > Hai can any one give me complete procedure for estimation of ACID > PHOSPHATASE, INVERTASE AND SUCROSE SYNTHASE with calculation formula. > Please dude I got procedures but I couldn't sort out how to arrive > mathematically.Here are references for those estimations Then you need to get a good book on enzymology and learn how to work this out, it is not rocket science. For example, if you release nitrophenol from p-nitrophenylphosphate by a phosphatase, the determination is by photometry. The absorbance is related to the concentration of NP (the product) by Lambert-Beer's law. Concentration times volume gives amount, amount by time gives activity. Activity divided by protein amount in the assay gives specific activity. In your case I would recommend: I.H. Segel: Biochemical Calculations, 2nd Ed. (Wiley) 1976, ISBN 978-0471774211 Don't be confused by the age of this book, maths hasn't changed much in the last 30 years! From stanleycheungkk from yahoo.com Sat May 16 00:55:05 2009 From: stanleycheungkk from yahoo.com (Stanley Cheung) Date: Sat May 16 13:31:32 2009 Subject: Monomeric Abeta detection in brain of APP tg mice using western blotting Message-ID: <836243.35962.qm@web54507.mail.re2.yahoo.com> Hi there, Does anyone have experiences in detecting and quantifying soluble monomeric Abeta in brain of APP transgenic mice (Tg2576). There is no clear description on the loading amount in journals. I tried many times and eventually found a paper described using 100ug to 250ug of brain homogenate in TBS after ultracenfriguation, 100k xg. However, I believe there is a limited total protein concentration which can be extracted using TBS during homogenization. Actually, 250ug sample means too large volume for loading?on mini-SDS-PAGE gel. If for detecting monomeric Abeta (~4KD), the volume of the loading sample should be very small for better resolution. I tried to use TCA precipitation to reduce the volume, but the monomeric Abeta disappeared and the?precipitate cannot be dissolved completely. Compared with samples without ultracentrifugation (using the sample TCA precipitation method) and?the pellet obtained after ultracentrifugation, monomeric Abeta can be seen in these samples on the?blot. It makes me to think that TCA precipitation causes too much protein loss for small peptide or the abundance of the peptide in the sample is too little. I should not use the whole brain for doing western blotting of Abeta, it consumes too much sample. I found most papers only use ELISA for soluble Abeta detection, or immunoprecipitate the Abeta from large amount of sample and load it on the gel for WB. However, these two methods are too expensive in my lab. Lyophilization doesn't work too, because it increases the salt content too much which distorts the bands in the gel and the lyophilized sample cannot be totally dissolved in reduced volume. Can anyone suggest the loading amount and ways to concentrate the sample for WB detection? Or is it practical to use WB for such detection? Thank you very much for your opinion in advance. Sincerely, Stanley PhD Candidate Department of Medicine and Therapeutics Faculty of Medicine Chinese University of Hong Kong Send instant messages to your online friends http://uk.messenger.yahoo.com From novalidaddress from nurfuerspam.de Sat May 16 16:59:53 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Sat May 16 20:10:02 2009 Subject: Monomeric Abeta detection in brain of APP tg mice using western blotting References: Message-ID: <4cdc8ed5-3806-408d-b7cd-4eb27f999b98@u10g2000vbd.googlegroups.com> Hi Stanley, for concentration, you might use a very low cut off mw dialysis membrane and reduce the volume by "dialysis" against high mw poly- ethyleneglycol. You also first might might separate monomeric Abeta with 10kD cut off spin column concentrator and the concentrate the flow through with a 1kd concentrator. In the latter step, you also may obtain some desalting when you dilute your sample with dd water before the concentration. Just some thoughts and good luck! Wo From nadmsa1 from yahoo.com Sun May 17 15:41:42 2009 From: nadmsa1 from yahoo.com (nada mohamed) Date: Sun May 17 18:39:09 2009 Subject: RNA labeling at 5' Message-ID: <288179.46934.qm@web34402.mail.mud.yahoo.com> Dear all, ? I am a PhD student and I am studying RNA-Protein interaction. I need a protocol to label my RNA at 5'end with biotin, I found some researches using megascript kit to incorporate biotin and others using Maxiscript I am confused which one is suitable ? and is there any alternative methods, also what is the criteria that must be taken in account for RNA biotin labeling (to be sure that my RNA is not affected by biotin incorporation),?I mean the size of RNA the amount of?labeled?nucleotides. Any help will be appreciated.? From Nikola.Wenta from nottingham.ac.uk Mon May 18 04:57:41 2009 From: Nikola.Wenta from nottingham.ac.uk (Nikola Wenta) Date: Mon May 18 11:25:31 2009 Subject: SUMO antibody problem References: <200905171705.n4HH5iG11119@net.bio.net> Message-ID: <814A9FCB0AD790489679BFBCE6CA5E55303116@VUIEXCHC.ad.nottingham.ac.uk> Hi all! Does anyone have tried to detect SUMOylated protein? I have performed in vitro SUMOylation as well as in vivo. In both cases, I was able to detect my protein and a higher molecular protein band, corresponding to presumably the SUMOylated protein with the protein-specific antibody. However, when I use a SUMO-specific antibody, I fail to detect the SUMOylated protein. Does anyone have any suggestions how to get the SUMO antibody working? Would it be advisable to perform other methods to detect SUMOylation, i.e. MS? Thank you for any suggestions! Niko This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation. From tfitzwater from somalogic.com Mon May 18 09:48:51 2009 From: tfitzwater from somalogic.com (Tim Fitzwater) Date: Mon May 18 11:25:46 2009 Subject: RNA labeling at 5' Message-ID: <771BF0D090CE73449F7495D31E0BA0D14E9733@ELM.sladmin.com> The authors were able to initiate T7 transcription with 4 mM "5'-biotin-spacer 9-G as the starting nucleotide." McNamara, J. O., D. Kolonias, et al. (2007). "Multivalent 4-1BB binding aptamers costimulate CD8 T cells and inhibit tumor growth in mice." J Clin Invest. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dop t=Citation&list_uids=18060045 Tim From ngeraci from gmail.com Mon May 18 11:54:51 2009 From: ngeraci from gmail.com (Nicholas Geraci) Date: Mon May 18 13:23:42 2009 Subject: RNA based FISH on frozen tissue samples Message-ID: <975ec7520905180954m5d84d118xc335138dff42e164@mail.gmail.com> Hello everyone, I was wondering if anyone had a protocol, or could point me in the direction of one, for fluorescent in situ hybridization (FISH) of RNA probes to gene transcripts on frozen tissue sections. I've found numerous references to this effect, but none that seem to precisely match what we need (we are looking for transcripts in muscle cells). If there were any other resources you folks could suggest, or advice and procedures from your own experiences, it would be most appreciated. -- Nicholas S. Geraci, MS Research Associate Molecular & Cellular Pathobiology Children's Memorial Research Center 2430 North Halsted Street Chicago, Illinois 60614 (773) 755-6399 lab (513) 505-5339 mobile From kumar from lifesensors.com Tue May 19 14:52:14 2009 From: kumar from lifesensors.com (Suresh Kumar) Date: Tue May 19 15:33:31 2009 Subject: SUMO antibody problem (Nikola Wenta) In-Reply-To: <200905181703.n4IH3Yp11568@net.bio.net> References: <200905181703.n4IH3Yp11568@net.bio.net> Message-ID: <899CFF650DAF1245801B4A694F0E8064695290@ls-sbs.ls.local> Hi Nikola, Which SUMO antibody you use to detect SUMOylation and what are your western conditions? There are three major types of SUMOs (SUMO1, SUMO2 and SUMO3). You may need to select the right antibody depending on which isoform of SUMO you used for your in vitro SUMOylation. Antibody concentration and incubation time can also be critical. There are also Pan speficic antibodies that are supposed to recognize all three isoforms. Another method to detect sumoylation is to use in vitro translated (S35 methionine labeled) target protein and detect the slow migrating (sumoylated) target protein band using autoradiography. Since you can already detect SUMOylation using your protein antibody, it's your call if you want to use radio activity. Otherwise all you need is to get a good clean SUMO antibody and work out the conditions. Suresh _________________________ This e-mail is from Progenra Inc. The e-mail and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. Any unauthorized dissemination or copying of this e-mail or its attachments, and any use or disclosure of any information contained in them, is strictly prohibited and may be illegal. If you have received this e-mail in error, please notify or telephone 610-644-6974 and delete it from your system -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of methods-request@oat.bio.indiana.edu Sent: Monday, May 18, 2009 1:04 PM To: methods@magpie.bio.indiana.edu Subject: Methods Digest, Vol 48, Issue 15 Send Methods mailing list submissions to methods@net.bio.net To subscribe or unsubscribe via the World Wide Web, visit http://www.bio.net/biomail/listinfo/methods or, via email, send a message with subject or body 'help' to methods-request@net.bio.net You can reach the person managing the list at methods-owner@net.bio.net When replying, please edit your Subject line so it is more specific than "Re: Contents of Methods digest..." Today's Topics: 1. RNA labeling at 5' (nada mohamed) 2. SUMO antibody problem (Nikola Wenta) 3. RNA labeling at 5' (Tim Fitzwater) ---------------------------------------------------------------------- Message: 1 Date: Sun, 17 May 2009 13:41:42 -0700 (PDT) From: nada mohamed Subject: RNA labeling at 5' To: methods@magpie.bio.indiana.edu Message-ID: <288179.46934.qm@web34402.mail.mud.yahoo.com> Content-Type: text/plain; charset=iso-8859-1 Dear all, ? I am a PhD student and I am studying RNA-Protein interaction. I need a protocol to label my RNA at 5'end with biotin, I found some researches using megascript kit to incorporate biotin and others using Maxiscript I am confused which one is suitable ? and is there any alternative methods, also what is the criteria that must be taken in account for RNA biotin labeling (to be sure that my RNA is not affected by biotin incorporation),?I mean the size of RNA the amount of?labeled?nucleotides. Any help will be appreciated.? ------------------------------ Message: 2 Date: Mon, 18 May 2009 10:57:41 +0100 From: Nikola Wenta Subject: SUMO antibody problem To: Message-ID: <814A9FCB0AD790489679BFBCE6CA5E55303116@VUIEXCHC.ad.nottingham.ac.uk> Content-Type: text/plain; charset="iso-8859-1" Hi all! Does anyone have tried to detect SUMOylated protein? I have performed in vitro SUMOylation as well as in vivo. In both cases, I was able to detect my protein and a higher molecular protein band, corresponding to presumably the SUMOylated protein with the protein-specific antibody. However, when I use a SUMO-specific antibody, I fail to detect the SUMOylated protein. Does anyone have any suggestions how to get the SUMO antibody working? Would it be advisable to perform other methods to detect SUMOylation, i.e. MS? Thank you for any suggestions! Niko This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation. ------------------------------ Message: 3 Date: Mon, 18 May 2009 08:48:51 -0600 From: "Tim Fitzwater" Subject: RNA labeling at 5' To: Message-ID: <771BF0D090CE73449F7495D31E0BA0D14E9733@ELM.sladmin.com> Content-Type: text/plain; charset="us-ascii" The authors were able to initiate T7 transcription with 4 mM "5'-biotin-spacer 9-G as the starting nucleotide." McNamara, J. O., D. Kolonias, et al. (2007). "Multivalent 4-1BB binding aptamers costimulate CD8 T cells and inhibit tumor growth in mice." J Clin Invest. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dop t=Citation&list_uids=18060045 Tim ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 48, Issue 15 *************************************** From yansun2005 from gmail.com Wed May 20 15:06:15 2009 From: yansun2005 from gmail.com (yan sun) Date: Wed May 20 15:13:48 2009 Subject: 15% native gel Message-ID: Hi everybody, good afternoon. I was bothered by this 15% native gel shift experiment for a while. I study the interaction between NC protein and HIV TAR DNA. The gel was ordered from Bio-rad. It worked very well once. I ordered more, but this time there is no Complex band show up (interaction complex). I did not change anything, look like the gel has problem, just like denatured gel. But my friends used my gel, of course study his NC with HIV1 stem loop3 (different from my Tar DNA),his buffer is different from mine. The complex band shows up. Which means the gel itself did it's job. I felt quite confused. I made new running buffer (1X TBE), fresh DNA, fresh NC, new loading buffer(50 mM hepes,pH 7.5). Change one by one reaction condition each time, but none of them got me good results. My friends used sodium phosphoate buffer, 150mM NaCl. Actually it will break the nonspecific binding more than my buffer, right? How come he got reaction complex, but my is none? Since we used different Nucleic acids, of course the Kd values are different, there is nothing to compare. I am very very upset, could anybody experienced in this field give me some thoughts?? Thank you so much! Best, Frances -- Y. F.S. From gmaubach from ibn.a-star.edu.sg Wed May 20 19:11:05 2009 From: gmaubach from ibn.a-star.edu.sg (Gunter Maubach) Date: Wed May 20 21:32:01 2009 Subject: RNA isolation failed Message-ID: <298910B09CBCC849888776CECE9848ED147B40@E2K3.surromed.com.sg> Dear All, I recently did a liver isolation from mice after PBS and PFA (4%) perfusion. Because I had a number of mice I placed the tubes with the liver pieces into dry ice. I tried to isolate the total RNA using the kit we usually use (M&N RNAII). First I thawed the tissue piece in the lysis buffer which comes with the kit and did the homogenization using a polytron. Except for one sample (around 50ng/ml) I got something like 3.8ng/ml and so on. Next I tried the liquid nitrogen to disrupt the tissue, same result. I used always new liver pieces (around 0.5cm2) stored at -80grdC. I diluted the lysed tissue in case I had to much total RNA and therefore got the low yield due to problems with the column. Another of my colleagues performed the isolation, same result. The kit works fine with cells and others have used it with tissue without problems. I'm lost would could be the reason for these results and how to solve it. I can not imagine that the lysis is so poor. I would very much like to hear your opinions on this problem. Thanks in advance Gunter ______________________ !!! !!!!!!!!! !!!!!! !!! !!! !!! !!! !!! !!! !!! !!! !!!!!!!!! !!! !!! !!! !!! !!! !!! !!! !!! !!! !!! !!!!!!!!! !!! !!!!!! Dr. Gunter Maubach Institute of Bioengineering and Nanotechnology 31 Biopolis Way The Nanos #04-01 Singapore 138669 Tel: +65 6824-7142 Fax: +65 6478-9565 gmaubach@ibn.a-star.edu.sg This email is confidential and may be privileged. If you are not the intended recipient, please notify the sender and delete it. Please do not copy or use it for any purpose, or disclose its contents to any other person as it may be an offence under the Official Secrets Act. Thank you. Institute of Bioengineering and Nanotechnology http://www.ibn.a-star.edu.sg From usiva from ufl.edu Wed May 20 16:59:28 2009 From: usiva from ufl.edu (SIVAKUMAR) Date: Wed May 20 21:32:09 2009 Subject: Glyco stain Message-ID: <595350105.9301242856768147.JavaMail.osg@osgjas01.cns.ufl.edu> Hi Greetings I have been working on archael protein. Does any one could help me to figure out the problem in glycosylation staining with Proq emerald 300. I am getting non specific staining that means it stains even nonglycosylated protein which was run as negetive control on SDS-Page 14% gel. Even with the candy cane marker, nonglycosylated protein gives signal. Could any share your experineses. Thans, Siva From conferences from embl.de Wed May 20 19:24:07 2009 From: conferences from embl.de (EMBL Courses & Conferences) Date: Wed May 20 21:32:14 2009 Subject: Last Announcement: EMBO Conference Series on Protein Synthesis and Translational Control Message-ID: <200905210022.n4L0M7p13572@net.bio.net> Dear Colleagues, EMBO Conference Series on Protein Synthesis and Translational Control Partnered with the "CSHL Meeting on Translational Control" EMBL Heidelberg, Germany, 9 - 13 September 2009 This is to alert you of the upcoming registration and abstract deadline 1 June 2009. There will be no deadline extension possible, because the organizers meet soon after the deadline to discuss the session allocations of the submitted abstracts. From the registrations that we have had already and based on the success of the 2005 and 2007 meetings here in Heidelberg, this will be a very popular meeting for which we will need to limit participation to a maximum of 300 attendees. The time of registration will be an admission criterium in case that the meeting is oversubscribed. We invite you to register and submit your abstract as soon as possible via http://www.embl.de/conferences/TransControl/2009 Looking forward to welcoming you in September! Anne, Marina, Nahum and Matt For further inquiries, please contact: EMBL Course and Conference Office Bettina Schaefer Meyerhofstr. 1 69117 Heidelberg email: schaefer@embl.de phone: +49-6221-387-8836 If you do not wish to receive any further information about events taking place at EMBL, please reply with "unsubscribe" in the subject. From yansun2005 from gmail.com Wed May 20 20:22:37 2009 From: yansun2005 from gmail.com (yan sun) Date: Wed May 20 21:32:38 2009 Subject: 15% native gel shift expt. Message-ID: Hi everybody, good evening!! I was bothered by this 15% native gel shift experiment for a while. I study the interaction between NC protein and HIV TAR DNA. The gel was ordered from Bio-rad. It worked very well once. I ordered more, but this time there is no Complex band show up (interaction complex). I did not change anything, look like the gel has problem, just like denatured gel. But my friends used my gel, of course study his NC with HIV1 stem loop3 (different from my Tar DNA),his buffer is different from mine. The complex band shows up. Which means the gel itself did it's job. I felt quite confused. I made new running buffer (1X TBE), fresh DNA, fresh NC, new loading buffer(50 mM hepes,pH 7.5). Change one by one reaction condition each time, but none of them got me good results. My friends used sodium phosphoate buffer, 150mM NaCl. Actually it will break the nonspecific binding more than my buffer, right? How come he got reaction complex, but my is none? Since we used different Nucleic acids, of course the Kd values are different, there is nothing to compare. I am very very upset, could anybody experienced in this field give me some thoughts?? Thank you so much! -- Y. F.S. From lautys from gmail.com Wed May 20 21:42:00 2009 From: lautys from gmail.com (lautys) Date: Wed May 20 23:03:47 2009 Subject: 15% native gel shift expt. In-Reply-To: References: Message-ID: hi Yan Sun, I'm not an expert in this but putting me in your condition, I would to try out using my friend's protocol, including using the sodium phosphoate buffer. As a researcher, u know sometime things just work. Another alternative is consult Bio-rad's (not sales representative) technical group, they might able to troubleshoot your problem. If they product worked once for you, there is no reason why it does work anymore. wish you good luck. regards, Lau On Thu, May 21, 2009 at 9:22 AM, yan sun wrote: > Hi everybody, good evening!! > > I was bothered by this 15% native gel shift experiment for a while. > > I study the interaction between NC protein and HIV TAR DNA. > > The gel was ordered from Bio-rad. It worked very well once. I ordered more, > but this time there is no Complex band show up (interaction complex). > > I did not change anything, look like the gel has problem, just like > denatured gel. But my friends used my gel, of course study his NC with HIV1 > stem loop3 (different from my Tar DNA),his buffer is different from mine. > The complex band shows up. Which means the gel itself did it's job. > > I felt quite confused. I made new running buffer (1X TBE), fresh DNA, fresh > NC, new loading buffer(50 mM hepes,pH 7.5). Change one by one reaction > condition each time, but none of them got me good results. > > My friends used sodium phosphoate buffer, 150mM NaCl. Actually it will > break > the nonspecific binding more than my buffer, right? How come he got > reaction > complex, but my is none? Since we used different Nucleic acids, of course > the Kd values are different, there is nothing to compare. > > > > I am very very upset, could anybody experienced in this field give me some > thoughts?? > Thank you so much! > > -- > Y. F.S. > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From yansun2005 from gmail.com Wed May 20 22:04:25 2009 From: yansun2005 from gmail.com (yan sun) Date: Wed May 20 23:03:56 2009 Subject: 15% native gel shift expt. In-Reply-To: References: Message-ID: Hi Lau, 1) Thank you for your suggestion. I might try my friend's protocol. 2) I did consult with bio-rad's technical group. They said the native gel is not designed for gel shift assay. They suggested me to use 0.5XTBE instead of 1X TBE. They said high ionic strength might disturb the binding. I tried 0.5 X TBE, It did not work neither. Yeah, it was pretty confused. It is kind of ~{!1~}luck" thing, all of sudden, you could not repeat what you did, even did well many times before. Best,Yan On Wed, May 20, 2009 at 10:42 PM, lautys wrote: > hi Yan Sun, > > I'm not an expert in this but putting me in your condition, I would to try > out using my friend's protocol, including using the sodium phosphoate > buffer. As a researcher, u know sometime things just work. > > Another alternative is consult Bio-rad's (not sales representative) > technical group, they might able to troubleshoot your problem. If they > product worked once for you, there is no reason why it does work anymore. > > wish you good luck. > > regards, > Lau > > On Thu, May 21, 2009 at 9:22 AM, yan sun wrote: > >> Hi everybody, good evening!! >> >> I was bothered by this 15% native gel shift experiment for a while. >> >> I study the interaction between NC protein and HIV TAR DNA. >> >> The gel was ordered from Bio-rad. It worked very well once. I ordered >> more, >> but this time there is no Complex band show up (interaction complex). >> >> I did not change anything, look like the gel has problem, just like >> denatured gel. But my friends used my gel, of course study his NC with >> HIV1 >> stem loop3 (different from my Tar DNA),his buffer is different from mine. >> The complex band shows up. Which means the gel itself did it's job. >> >> I felt quite confused. I made new running buffer (1X TBE), fresh DNA, >> fresh >> NC, new loading buffer(50 mM hepes,pH 7.5). Change one by one reaction >> condition each time, but none of them got me good results. >> >> My friends used sodium phosphoate buffer, 150mM NaCl. Actually it will >> break >> the nonspecific binding more than my buffer, right? How come he got >> reaction >> complex, but my is none? Since we used different Nucleic acids, of course >> the Kd values are different, there is nothing to compare. >> >> >> >> I am very very upset, could anybody experienced in this field give me some >> thoughts?? >> Thank you so much! >> >> -- >> Y. F.S. >> _______________________________________________ >> Methods mailing list >> Methods@net.bio.net >> http://www.bio.net/biomail/listinfo/methods >> > > -- Y. F.S. From lautys from gmail.com Wed May 20 22:25:29 2009 From: lautys from gmail.com (lautys) Date: Wed May 20 23:04:02 2009 Subject: 15% native gel shift expt. In-Reply-To: References: Message-ID: haha! yea, same thing with me, i always have first trial run very well and struggle for the subsequent run... anyway, if what u bought is not designed for gel shift assay, u might what to switch to a proper product to ease things out. cheer~ Lau On Thu, May 21, 2009 at 11:04 AM, yan sun wrote: > Hi Lau, > > 1) Thank you for your suggestion. I might try my friend's protocol. > > 2) I did consult with bio-rad's technical group. They said the native gel > is not designed for gel shift assay. They suggested me to use 0.5XTBE > instead of 1X TBE. They said high ionic strength might disturb the binding. > I tried 0.5 X TBE, It did not work neither. > > Yeah, it was pretty confused. It is kind of ~{!1~}luck" thing, all of > sudden, you could not repeat what you did, even did well many times > before. > > > > Best,Yan > > > > > On Wed, May 20, 2009 at 10:42 PM, lautys wrote: > >> hi Yan Sun, >> >> I'm not an expert in this but putting me in your condition, I would to try >> out using my friend's protocol, including using the sodium phosphoate >> buffer. As a researcher, u know sometime things just work. >> >> Another alternative is consult Bio-rad's (not sales representative) >> technical group, they might able to troubleshoot your problem. If they >> product worked once for you, there is no reason why it does work anymore. >> >> wish you good luck. >> >> regards, >> Lau >> >> On Thu, May 21, 2009 at 9:22 AM, yan sun wrote: >> >>> Hi everybody, good evening!! >>> >>> I was bothered by this 15% native gel shift experiment for a while. >>> >>> I study the interaction between NC protein and HIV TAR DNA. >>> >>> The gel was ordered from Bio-rad. It worked very well once. I ordered >>> more, >>> but this time there is no Complex band show up (interaction complex). >>> >>> I did not change anything, look like the gel has problem, just like >>> denatured gel. But my friends used my gel, of course study his NC with >>> HIV1 >>> stem loop3 (different from my Tar DNA),his buffer is different from mine. >>> The complex band shows up. Which means the gel itself did it's job. >>> >>> I felt quite confused. I made new running buffer (1X TBE), fresh DNA, >>> fresh >>> NC, new loading buffer(50 mM hepes,pH 7.5). Change one by one reaction >>> condition each time, but none of them got me good results. >>> >>> My friends used sodium phosphoate buffer, 150mM NaCl. Actually it will >>> break >>> the nonspecific binding more than my buffer, right? How come he got >>> reaction >>> complex, but my is none? Since we used different Nucleic acids, of course >>> the Kd values are different, there is nothing to compare. >>> >>> >>> >>> I am very very upset, could anybody experienced in this field give me >>> some >>> thoughts?? >>> Thank you so much! >>> >>> -- >>> Y. F.S. >>> _______________________________________________ >>> Methods mailing list >>> Methods@net.bio.net >>> http://www.bio.net/biomail/listinfo/methods >>> >> >> > > > -- > Y. F.S. > From rahimpour_a from yahoo.com Thu May 21 14:50:52 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Fri May 22 12:23:16 2009 Subject: cho DHFR Message-ID: <477301.83909.qm@web52707.mail.re2.yahoo.com> Hi ? are ?there CHO DHFR (-) cells other than dg44 and duxb11? ? Does anybody know commercial supliers of CHO DHFR (-) cells other than invitrogen? From engelbert_buxbaum from hotmail.com Fri May 22 13:06:49 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri May 22 19:29:40 2009 Subject: Glyco stain References: Message-ID: Am 20.05.2009, 17:59 Uhr, schrieb SIVAKUMAR : > Hi > Greetings > I have been working on archael protein. Does any one could help me to > figure out the problem in glycosylation staining with Proq emerald 300. > I am getting non specific staining that means it stains even > nonglycosylated protein which was run as negetive control on SDS-Page > 14% gel. Even with the candy cane marker, nonglycosylated protein gives > signal. Could any share your experineses. Have you tried the classical PAS-staining, just as a sanity check? Are you satining in the gels directly or on blots? From Vince.Mulholland from sasa.gsi.gov.uk Tue May 26 06:10:13 2009 From: Vince.Mulholland from sasa.gsi.gov.uk (Vince Mulholland) Date: Tue May 26 14:44:07 2009 Subject: molecular biology textbooks Message-ID: My favourite of the newer books is Current Protocols Essential Laboratory Techniques (Sean Gallagher, Emily A. Wiley) ISBN: 978-0-470-08993-4 Paperback, 806 pages. Published by Wiley. It is not too expensive & it is quite an interesting read, with a lot of background for a "manual". Regards, Vince Mulholland Correspondents should note that all communications to or from SASA (Science and Advice for Scottish Agriculture – a Division of the Rural Payments and Inspections Directorate of the Scottish Government) may be automatically logged, monitored and/or recorded for lawful purposes. The original of this email was scanned for viruses by the Government Secure Intranet (GSi) virus scanning service. On leaving the GSi this email was certified virus-free. From rahimpour_a from yahoo.com Tue May 26 11:50:39 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Tue May 26 14:44:19 2009 Subject: expression of a subunit Message-ID: <496861.42412.qm@web52704.mail.re2.yahoo.com> hi I am going to express a mutant subunit of a multimeric complex protein in mammalian cells , this subunit is expected to incorporate in the whole protein complex, I dont know if attachment of small tags like his tag or HA tag would disrupt its structure and prevent its incorporation in a complex. does any one has any experience? From celesteel from gmail.com Tue May 26 16:05:59 2009 From: celesteel from gmail.com (L Celeste) Date: Tue May 26 16:38:09 2009 Subject: Help with PCR reaction Phusion Flash - wrong sized fragment Message-ID: <7f3233410905261405k127bdc41u4c813783d0c424c7@mail.gmail.com> Hi everyone, I am working on PCR amplification of a large fragment containing a gene with an expected amplicon of 11 kb. We have been using Phusion Flash Master Mix and been getting a bright 2 kb band and a few other faint non-specific bands. We have sent the primers and the DNA template for sequencing. Since there is a limitation on how large a fragment one could sequence, we only know that each primer was able to correctly amplify the first 1 kb from both end of the target 11 kb DNA. We then use another set of primers (Let's call it B (+) & (-) )that bind to areas near the middle of the 11 kb target fragment with the first set of primers (Let's call it A (+) & (-) ) to see if we were able to amplife an approximately 5.5 kb fragment. So with A (-) & B(+) primers, we got a clear 6 kb band. But with A (+) & B (-), we not only got the expected 6 kb band, but also the often-seen 2 kb band and a few other non-specific bands. So, we thought primer A (+) was not specific enough and tried to redesign the primers, but the new A (+) primer still give the same result as before. Could anyone please provide some advice on what to do next or what could have gone wrong? Some extra information: I have also done a negative control, and there was no band. Thanks Celeste From celesteel from gmail.com Tue May 26 16:15:35 2009 From: celesteel from gmail.com (L Celeste) Date: Tue May 26 16:38:22 2009 Subject: PCR problem: pfu working selectively? Message-ID: <7f3233410905261415i5cd41e70qd861b4c910f04001@mail.gmail.com> Hi everyone, I have been trying to amplify a 9 kb pBAC fragment with Phusion Flash Master Mix. Although we didn't get the right bands, but we got bands (one at 7 kb) at annealing temperature of up to 60oC and extension temperature of 72oC. But then when we switched to Pfu with annealing temperature of 60 oC and 61 oC, and extension temperature of 68 oC we were not able to produce any bands at all. Thinking that our Pfu might not be working at all, I repeated this experiment along with another experiment (target fragement 11 kb) at 55 oC. We got bands for the experiment with target fragment of 11 kb, but again no band for the orginal pBAC experiment. Any advice would be appreciated. Thanks Celeste From taliaferroD from mail.nih.gov Tue May 26 17:18:17 2009 From: taliaferroD from mail.nih.gov (Dwayne Taliaferro) Date: Tue May 26 18:04:52 2009 Subject: Help with PCR reaction Phusion Flash - wrong sized fragment In-Reply-To: <7f3233410905261405k127bdc41u4c813783d0c424c7@mail.gmail.com> Message-ID: There are three possible scenarios. They're somewhat exotic and I'm sure someone on here will suggest a more pragmatic cause/solution 1. The 2kb fragment is nonspecific. Not likely since you say the 2kb fragment contains 1kb from each end of the target. 2. It is likely you are amplifying a version of the gene with a deletion in it. Possible since it is a smaller amplicon, it would out compete the larger product. You may see the larger product if you increase the number of cycles. 3. There's significant structure in the ends being amplified such that they effectively "looping out" part of the sequence or stopping the polymerase. These truncated products have enough overlap to amplify each other to produce a 2kb product. Don't think this is likely. Sounds crazy. What I would do: A. Sequence the 6kb products to be sure they are correct. B. Excise the two 6kb fragments and use them in a PCR reaction to see if you can generate the 11kB amplicon. C. If B doesn't work, increase the cycles on the original PCR to see if the 11kb product shows up. If it does, cut it out and use it in another reaction. On 5/26/09 5:05 PM, "L Celeste" wrote: > Hi everyone, > > I am working on PCR amplification of a large fragment containing a gene with > an expected amplicon of 11 kb. We have been using Phusion Flash Master Mix > and been getting a bright 2 kb band and a few other faint non-specific > bands. We have sent the primers and the DNA template for sequencing. Since > there is a limitation on how large a fragment one could sequence, we only > know that each primer was able to correctly amplify the first 1 kb from both > end of the target 11 kb DNA. > > We then use another set of primers (Let's call it B (+) & (-) )that bind to > areas near the middle of the 11 kb target fragment with the first set of > primers (Let's call it A (+) & (-) ) to see if we were able to amplife an > approximately 5.5 kb fragment. So with A (-) & B(+) primers, we got a clear > 6 kb band. But with A (+) & B (-), we not only got the expected 6 kb band, > but also the often-seen 2 kb band and a few other non-specific bands. So, we > thought primer A (+) was not specific enough and tried to redesign the > primers, but the new A (+) primer still give the same result as before. > > Could anyone please provide some advice on what to do next or what could > have gone wrong? > > Some extra information: > I have also done a negative control, and there was no band. > > Thanks > Celeste > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods From celesteel from gmail.com Tue May 26 18:01:24 2009 From: celesteel from gmail.com (L Celeste) Date: Tue May 26 19:50:57 2009 Subject: Help with PCR reaction Phusion Flash - wrong sized fragment Message-ID: <7f3233410905261601o68ca9c78i84defadb506c949b@mail.gmail.com> 1. When we did sequencing, we only send the DNA template as well as the primer, we didn't actually sequence the 2 kb fragment (and it actually is more like 2.5 kb). So our assumption is that that 2 kb fragment we are seeing constantly does contain the 1 kb from the sequencing reaction. 2. What number of cycles would you suggest? I have tried 25, 27 and 30 cycles. Thanks From pjie2 from cam.ac.uk Tue May 26 19:14:52 2009 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Tue May 26 19:51:05 2009 Subject: Help with PCR reaction Phusion Flash - wrong sized fragment In-Reply-To: References: Message-ID: <783f0aF1k93q4U1@mid.individual.net> Dwayne Taliaferro wrote: > There are three possible scenarios. They're somewhat exotic and I'm sure > someone on here will suggest a more pragmatic cause/solution > > 1. The 2kb fragment is nonspecific. Not likely since you say the 2kb > fragment contains 1kb from each end of the target. > > 2. It is likely you are amplifying a version of the gene with a deletion in > it. Possible since it is a smaller amplicon, it would out compete the > larger product. You may see the larger product if you increase the number > of cycles. > > 3. There's significant structure in the ends being amplified such that they > effectively "looping out" part of the sequence or stopping the polymerase. > These truncated products have enough overlap to amplify each other to > produce a 2kb product. Don't think this is likely. Sounds crazy. Not necessarily crazy. If there's a direct repeat within the amplicon you're trying to amplify, then partial products from each end might be able to anneal to one another and "delete" the section between the repeat units. The smaller product would then outcompete the larger. In terms of how to fix it, I think the first priority is to get the full sequence of the 2kb band, so you can get a handle on how/why it's being generated. Since you can't get the full sequence using only A (+) and (-) primers, you'll need to design further sequencing primers inside those to get the sequence of the centre of the 2kb fragment. In regard to the reactions using primer set B, I don't see how the 2kb fragment from the A (+) / B (-) reaction can be the same as the 2kb fragment from the original reaction: you know the latter has both A (+) and A (-) binding sites or you wouldn't be able to sequence it. Sequencing this other 2kb band might also give you clues about what's going on. The suggestion of excising the two 6kb bands from the A/B reactions and then using them in an extension PCR is a good one, and may well let you get the full amplicon. Peter Ellis From editor from gene-quantification.info Thu May 28 05:58:07 2009 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Thu May 28 11:12:08 2009 Subject: qPCR NEWS May 2009 - focus on RNA integrity Message-ID: qPCR NEWS May 2009 - focus on RNA integrity ------------------------------------------------------------ Dear researcher, dear Gene Quantification page reader, Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene Quantification homepage. The focus of this newsletter issue is: - The MIQE Guidelines Minimum Information for Publication of Quantitative Real-Time PCR Experiments - RNA integrity - latest papers - PowerNest - illuminating error in qPCR experiment design - new TAG CLOUD for better navigation => http://directory.gene-quantification.info/ - New qPCR workshop modules at the TATAA Biocenter Germany => http://tataa.gene-quantification.info/ - European wide qPCR application workshops => register now ! -------------------------------------------------------------------------------- The MIQE Guidelines Minimum Information for Publication of Quantitative Real-Time PCR Experiments Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson, Jan Hellemans, Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W. Pfaffl, Gregory L. Shipley, Jo Vandesompele, & Carl T. Wittwer Clinical Chemistry 2009, 55(4): 611-622 => http://www.gene-quantification.de/miqe-bustin-et-al-clin-chem-2009.pdf BACKGROUND: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments. CONTENT: The Minimum Information for Publication of Quantitative Real- Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. SUMMARY: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results. => http://miqe.gene-quantification.info/ -------------------------------------------------------------------------------- As part of the MIQE guidelines - chapter 5.1 - the RNA quality control is an essential step in the quantification process. 5. Nucleic acid quality control 5.1. RNA samples Since it is advisable to use approximately the same amount of RNA for cDNA synthesis when comparing different samples, quantification of RNA in extracted samples is important. However, there are several procedures in common use, including spectrophotometry (Nanodrop), microfluidic analysis (Agilent BioAnalyser, BioRad Experion), capillary gel electrophoresis (Qiagen QIAexcel) or fluorescent dye detection (Ribogreen); all produce different results making it unwise to compare data obtained using the different methods. The preferred method for RNA quantity determination uses fluorescent RNA binding dyes, for example RiboGreen, which are best for the detection of low target concentrations. In any case, it is advisable to measure all samples using one method only and to report this information. ... ... ... latest papers => http://www.gene-quantification.de/rna-integrity2.html - Reverse transcription-quantitative polymerase chain reaction: description of a RIN-based algorithm for accurate data normalization. - Time course analysis of RNA stability in human placenta. - Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation. - A comparison and evaluation of RNA quantification methods using viral, prokaryotic, and eukaryotic RNA over a 10(4) concentration range. - Validation of lab-on-chip capillary electrophoresis systems for total RNA quality and quantity control. - Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials. - Optimization of the method of RNA isolation from paraffin blocks to assess gene expression in breast cancer. - Measuring microRNAs: comparisons of microarray and quantitative PCR measurements, and of different total RNA prep methods. - Focus on RNA isolation: obtaining RNA for microRNA (miRNA) expression profiling analyses of neural tissue. - Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. - Stability of RNA isolated from post-mortem tissues of Atlantic salmon (Salmo salar L.) - Prediction of qualitative outcome of oligonucleotide microarray hybridization by measurement of RNA integrity using the 2100 Bioanalyzer capillary electrophoresis system. - Removal of contaminating DNA from commercial nucleic acid extraction kit reagents. -------------------------------------------------------------------------------- PowerNest - illuminating error in qPCR experiment design PowerNest is a software tool enabling experimenters to explore the effect of sampling on noise propagation throughout qPCR assays. The sampling process is assumed to be comprised of a number of levels; the acquisition of a sample and the preparation of extracted material, reverse-transcription of the mRNA, and the qPCR itself. Given a small set of data, representative of a larger assay, the error at each stage of the experiment is profiled using a nested-ANOVA. Armed with this information, PowerNest allows the experimenter to explore the effects of modifications to the experimental design on the expected total error of the assay. When given the financial cost of replicates at each level, PowerNest will calculate a cost-optimal sampling-plan, delivering an experiment design that will minimise processing error and maximise the statistical resolution of the assay. The software is temporarily undergoing final testing, during which time it has been made available as a free download => http://www.gene-quantification.de/main-bioinf.shtml#powernest PowerNest Poster => http://www.gene-quantification.de/powernest-poster.pdf -------------------------------------------------------------------------------- Upcoming Events World-wide academic and commercial qPCR Events http://events.gene-quantification.info/ Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars, qPCR Education Program, etc. Please submit your qPCR event here => events@gene- quantification.info -------------------------------------------------------------------------------- qPCR WORKSHOP BioEPS GmbH / TATAA Biocenter Germany - qPCR Application workshops At the TATAA Biocenter Germany we offer qPCR application workshops, a 3-day qPCR Core Module and a 2-day qPCR Biostatistics Module. All courses are held regularly in G?teborg, Sweden, in English and in Freising-Weihenstephan, Germany, in German and English, and in Prague, Czech Republic in English and Czech. Depending on the occasion the workshop language and the different prices may apply. Further customized workshops and specialized trainings will be held as well across Europe and world-wide. TATAA Biocenter Germany workshops are held in cooperation with BioEPS GmbH, located at the campus of the Technical University of Munich, in Freising-Weihenstephan, very close to the Munich Airport (MUC). For more information and registration, please see our web page: => http://TATAA.gene-quantification.info/ Course Occasions 2009: 3-day qPCR Core Module (Mon. - Wed.) 2-day BioStatistics Module (Thu. - Fri.) 3-day single-cell qPCR Module (Mon. - Wed.) 3-day microRNA Module (Mon. - Wed.) 15 - 19 Juni 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics (Thu. - Fri.) 13 - 15 Juli 2009 (E) NEW microRNA qPCR (in cooperation with Qiagen) 27 - 31 Juli 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics (Thu. - Fri.) 14 - 16 September 2009 (E) NEW single-cell qPCR 19 - 23 September 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2- day BioStatistics (Thu. - Fri.) 26 - 28 October 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) 16 - 20 November 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2- day BioStatistics (Thu. - Fri.) 7 - 11 December 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2- day BioStatistics (Thu. - Fri.) => http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pdf => http://www.gene-quantification.de/bioeps-courses-autumn-2009.pdf => http://www.gene-quantification.de/single-cell-qpcr-course-sept-2009.pdf -------------------------------------------------------------------------------- Forward Please send the qPCR NEWS to further scientists and friends who are interested in qPCR ! Best regards, Michael W. Pfaffl responsible Editor of the Gene Quantification Pages http://www.gene-quantification.info -------------------------------------------------------------------------------- If this newsletter is not displayed correctly by your email client, please use following link: http://qpcrnews.gene-quantification.info/ The qPCR NEWS and the Gene Quantification Pages are educational sites with the only purpose of facilitating access to qPCR related information on the internet. The qPCR NEWS and the Gene Quantification Pages are edited by Michael W. Pfaffl. Copyright ? 2005 - 2009 All rights reserved. Any unauthorized use, reproduction, or transfer of this message or its contents, in any medium, is strictly prohibited. Disclaimer & Copyrights are displayed on the homepage www.gene-quantification.com To subscribe or change your e-mail address in qPCR NEWS, and if you would like to receive future issues FREE of charge, please send an e- mail with the subject SUBSCRIBE to mailto:newsletter@gene- quantification.info?subject=SUBSCRIBE From saxena from nus.edu.sg Thu May 28 08:59:16 2009 From: saxena from nus.edu.sg (Gourvendu Saxena) Date: Thu May 28 11:12:17 2009 Subject: EDTA solution becomes slurry Message-ID: Dear All I am trying to prepare 0.5 M EDTA solution pH 8. I used 10 N NaOH for = adjusting the pH but every time it becomes a white semisolid slurry = before reaching the desired pH. Please forward your suggestions. Thanks Gourvendu From hroychow from nmsu.edu Thu May 28 12:38:37 2009 From: hroychow from nmsu.edu (hroychow@nmsu.edu) Date: Thu May 28 16:32:00 2009 Subject: EDTA solution becomes slurry In-Reply-To: References: Message-ID: Are you talking about "slurry" or whitish ppt? Slurry is quite serious, and you may have a bad batch of EDTA. However, if you are unable to dissolve the EDTA at 0.5M with the NaOH you have, it may be the NaOH it self. Make a fresh solution. BTW, why use 10N? If you are making a lot, I would suggest use pellets instead (calculate the amt you may need and use slightly less than that. ----- Original Message ----- From: Gourvendu Saxena Date: Thursday, May 28, 2009 10:19 am Subject: EDTA solution becomes slurry To: methods@magpie.bio.indiana.edu > > Dear All > I am trying to prepare 0.5 M EDTA solution pH 8. I used 10 N > NaOH for > > djusting the pH but every time it becomes a white semisolid slurry > efore reaching the desired pH. Please forward your suggestions. > Thanks > Gourvendu > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From scedberg from aol.com Thu May 28 15:12:33 2009 From: scedberg from aol.com (stephen edberg) Date: Thu May 28 16:32:09 2009 Subject: Benzoic Acid Assay Message-ID: <923B6C44-5611-40BC-978A-D93798EF518B@aol.com> Do you have a simple assay for benzoic acid. Prof. Stephen Edberg From virashkgupta from gmail.com Thu May 28 23:11:07 2009 From: virashkgupta from gmail.com (Virash Gupta) Date: Thu May 28 23:44:25 2009 Subject: Methods Digest, Vol 48, Issue 21-EDTA solution becomes slurry Message-ID: Use equivalent amount of solid NaOH pellets (one by one) instead of solution while required amount of Tris.Cl in water is being continuousely stirred on a megnetic stirrer with pH meter electrode dipped in. On 5/28/09, methods-request@oat.bio.indiana.edu < methods-request@oat.bio.indiana.edu> wrote: > > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. Re: expression of a subunit (DK) > 2. qPCR NEWS May 2009 - focus on RNA integrity > (Editor www.Gene-Quantification.info) > 3. EDTA solution becomes slurry (Gourvendu Saxena) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 27 May 2009 19:46:43 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: expression of a subunit > To: methods@net.bio.net > Message-ID: > > In article , Azam > Rahimpour wrote: > >hi > >I am going to express a mutant subunit of a multimeric complex protein in > > mammalian cells , this subunit is expected to incorporate in the whole > protein > > complex, I dont know if attachment of small tags like his tag or HA tag > would > > disrupt its structure and prevent its incorporation in a complex. does > any one > > has any experience? > > It's protein dependent. Sometimes it does and sometimes it doesn't. > You'll have to try and find out. Try both N- and C-terminal tags. > > > > ------------------------------ > > Message: 2 > Date: Thu, 28 May 2009 03:58:07 -0700 (PDT) > From: "Editor www.Gene-Quantification.info" > > Subject: qPCR NEWS May 2009 - focus on RNA integrity > To: methods@net.bio.net > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > qPCR NEWS May 2009 - focus on RNA integrity > ------------------------------------------------------------ > > > > Dear researcher, > dear Gene Quantification page reader, > > Our newsletter informs about the latest news in quantitative real-time > PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene > Quantification homepage. The focus of this newsletter issue is: > > - The MIQE Guidelines Minimum Information for Publication of > Quantitative Real-Time PCR Experiments > - RNA integrity - latest papers > - PowerNest - illuminating error in qPCR experiment design > - new TAG CLOUD for better navigation => > http://directory.gene-quantification.info/ > - New qPCR workshop modules at the TATAA Biocenter Germany => > http://tataa.gene-quantification.info/ > - European wide qPCR application workshops => register now ! > > > > -------------------------------------------------------------------------------- > > The MIQE Guidelines Minimum Information for Publication of > Quantitative Real-Time PCR Experiments > > Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson, Jan Hellemans, > Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W. > Pfaffl, Gregory L. Shipley, Jo Vandesompele, & Carl T. Wittwer > Clinical Chemistry 2009, 55(4): 611-622 > > => http://www.gene-quantification.de/miqe-bustin-et-al-clin-chem-2009.pdf > > BACKGROUND: Currently, a lack of consensus exists on how best to > perform and interpret quantitative real-time PCR (qPCR) experiments. > The problem is exacerbated by a lack of sufficient experimental detail > in many publications, which impedes a reader's ability to evaluate > critically the quality of the results presented or to repeat the > experiments. > > CONTENT: The Minimum Information for Publication of Quantitative Real- > Time PCR Experiments (MIQE) guidelines target the reliability of > results to help ensure the integrity of the scientific literature, > promote consistency between laboratories, and increase experimental > transparency. MIQE is a set of guidelines that describe the minimum > information necessary for evaluating qPCR experiments. Included is a > checklist to accompany the initial submission of a manuscript to the > publisher. By providing all relevant experimental conditions and assay > characteristics, reviewers can assess the validity of the protocols > used. Full disclosure of all reagents, sequences, and analysis methods > is necessary to enable other investigators to reproduce results. MIQE > details should be published either in abbreviated form or as an online > supplement. > > SUMMARY: Following these guidelines will encourage better > experimental practice, allowing more reliable and unequivocal > interpretation of qPCR results. > > => http://miqe.gene-quantification.info/ > > > > -------------------------------------------------------------------------------- > > As part of the MIQE guidelines - chapter 5.1 - the RNA quality control > is an essential step in the quantification process. > > 5. Nucleic acid quality control > 5.1. RNA samples > Since it is advisable to use approximately the same amount of RNA for > cDNA synthesis when comparing different samples, quantification of RNA > in extracted samples is important. However, there are several > procedures in common use, including spectrophotometry (Nanodrop), > microfluidic analysis (Agilent BioAnalyser, BioRad Experion), > capillary gel electrophoresis (Qiagen QIAexcel) or fluorescent dye > detection (Ribogreen); all produce different results making it unwise > to compare data obtained using the different methods. The preferred > method for RNA quantity determination uses fluorescent RNA binding > dyes, for example RiboGreen, which are best for the detection of low > target concentrations. In any case, it is advisable to measure all > samples using one method only and to report this > information. ... ... ... > > latest papers => http://www.gene-quantification.de/rna-integrity2.html > > - Reverse transcription-quantitative polymerase chain reaction: > description of a RIN-based algorithm for accurate data normalization. > - Time course analysis of RNA stability in human placenta. > - Evaluation of isolation methods and RNA integrity for bacterial RNA > quantitation. > - A comparison and evaluation of RNA quantification methods using > viral, prokaryotic, and eukaryotic RNA over a 10(4) concentration > range. > - Validation of lab-on-chip capillary electrophoresis systems for > total RNA quality and quantity control. > - Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to > enhance expression analysis from formalin fixed paraffin embedded > (FFPE) materials. > - Optimization of the method of RNA isolation from paraffin blocks to > assess gene expression in breast cancer. > - Measuring microRNAs: comparisons of microarray and quantitative PCR > measurements, and of different total RNA prep methods. > - Focus on RNA isolation: obtaining RNA for microRNA (miRNA) > expression profiling analyses of neural tissue. > - Systematic analysis of microRNA expression of RNA extracted from > fresh frozen and formalin-fixed paraffin-embedded samples. > - Stability of RNA isolated from post-mortem tissues of Atlantic > salmon (Salmo salar L.) > - Prediction of qualitative outcome of oligonucleotide microarray > hybridization by measurement of RNA integrity using the 2100 > Bioanalyzer capillary electrophoresis system. > - Removal of contaminating DNA from commercial nucleic acid extraction > kit reagents. > > > > -------------------------------------------------------------------------------- > > PowerNest - illuminating error in qPCR experiment design > > PowerNest is a software tool enabling experimenters to explore the > effect of sampling on noise propagation throughout qPCR assays. The > sampling process is assumed to be comprised of a number of levels; the > acquisition of a sample and the preparation of extracted material, > reverse-transcription of the mRNA, and the qPCR itself. Given a small > set of data, representative of a larger assay, the error at each stage > of the experiment is profiled using a nested-ANOVA. > Armed with this information, PowerNest allows the experimenter to > explore the effects of modifications to the experimental design on the > expected total error of the assay. When given the financial cost of > replicates at each level, PowerNest will calculate a cost-optimal > sampling-plan, delivering an experiment design that will minimise > processing error and maximise the statistical resolution of the assay. > The software is temporarily undergoing final testing, during which > time it has been made available as a free download => > http://www.gene-quantification.de/main-bioinf.shtml#powernest > > PowerNest Poster => http://www.gene-quantification.de/powernest-poster.pdf > > > > -------------------------------------------------------------------------------- > > Upcoming Events World-wide academic and commercial qPCR Events > http://events.gene-quantification.info/ > > Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars, > qPCR Education Program, etc. > Please submit your qPCR event here => events@gene- > quantification.info > > > > -------------------------------------------------------------------------------- > > qPCR WORKSHOP > > BioEPS GmbH / TATAA Biocenter Germany - qPCR Application workshops > > At the TATAA Biocenter Germany we offer qPCR application workshops, a > 3-day qPCR Core Module and a 2-day qPCR Biostatistics Module. All > courses are held regularly in G?teborg, Sweden, in English and in > Freising-Weihenstephan, Germany, in German and English, and in Prague, > Czech Republic in English and Czech. > Depending on the occasion the workshop language and the different > prices may apply. Further customized workshops and specialized > trainings will be held as well across Europe and world-wide. > TATAA Biocenter Germany workshops are held in cooperation with BioEPS > GmbH, located at the campus of the Technical University of Munich, in > Freising-Weihenstephan, very close to the Munich Airport (MUC). For > more information and registration, please see our web page: > => http://TATAA.gene-quantification.info/ > > > Course Occasions 2009: > > 3-day qPCR Core Module (Mon. - Wed.) > 2-day BioStatistics Module (Thu. - Fri.) > 3-day single-cell qPCR Module (Mon. - Wed.) > 3-day microRNA Module (Mon. - Wed.) > > > 15 - 19 Juni 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day > BioStatistics (Thu. - Fri.) > 13 - 15 Juli 2009 (E) NEW microRNA qPCR (in cooperation with > Qiagen) > 27 - 31 Juli 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day > BioStatistics (Thu. - Fri.) > 14 - 16 September 2009 (E) NEW single-cell qPCR > 19 - 23 September 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2- > day BioStatistics (Thu. - Fri.) > 26 - 28 October 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) > 16 - 20 November 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2- > day BioStatistics (Thu. - Fri.) > 7 - 11 December 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2- > day BioStatistics (Thu. - Fri.) > > => http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pdf > => http://www.gene-quantification.de/bioeps-courses-autumn-2009.pdf > => http://www.gene-quantification.de/single-cell-qpcr-course-sept-2009.pdf > > > > -------------------------------------------------------------------------------- > > Forward Please send the qPCR NEWS to further scientists and friends > who are interested in qPCR ! > > > Best regards, > > Michael W. Pfaffl > responsible Editor of the Gene Quantification Pages > http://www.gene-quantification.info > > > > -------------------------------------------------------------------------------- > > If this newsletter is not displayed correctly by your email client, > please use following link: > http://qpcrnews.gene-quantification.info/ > > The qPCR NEWS and the Gene Quantification Pages are educational sites > with the only purpose of facilitating access to qPCR related > information on the internet. The qPCR NEWS and the Gene > Quantification Pages are edited by Michael W. Pfaffl. Copyright ? > 2005 - 2009 All rights reserved. Any unauthorized use, reproduction, > or transfer of this message or its contents, in any medium, is > strictly prohibited. Disclaimer & Copyrights are displayed on the > homepage www.gene-quantification.com > To subscribe or change your e-mail address in qPCR NEWS, and if you > would like to receive future issues FREE of charge, please send an e- > mail with the subject SUBSCRIBE to mailto:newsletter@gene- > quantification.info?subject=SUBSCRIBE > > > > > > > > ------------------------------ > > Message: 3 > Date: Thu, 28 May 2009 21:59:16 +0800 > From: "Gourvendu Saxena" > Subject: EDTA solution becomes slurry > To: > Message-ID: > > Content-Type: text/plain; charset="iso-8859-1" > > > Dear All > I am trying to prepare 0.5 M EDTA solution pH 8. I used 10 N NaOH for > = adjusting the pH but every time it becomes a white semisolid slurry > = before reaching the desired pH. Please forward your suggestions. > Thanks > Gourvendu > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 48, Issue 21 > *************************************** > -- Dr V K Gupta Sr Microbiologist (Molecular Biology) Insect Molecular Biology Lab Department of Entomology Punjab Agricultural University Ludhiana (Pb)-141004- India M: 09815963210 From srb10 from st-andrews.ac.uk Fri May 29 10:53:12 2009 From: srb10 from st-andrews.ac.uk (Simon Bushell) Date: Fri May 29 13:02:02 2009 Subject: Using Histidine as a Nickel Elution Buffer Message-ID: <4A2004E8.5080108@st-andrews.ac.uk> So I am purifying a protein complex using Ni-NTA chromatography. One of the subunits is his-tagged, its binding partner isn't, and they are able to co-purify from a nickel column. So far so good. However it seems that when imidazole concentrations get above 50mM, the complex soon comes apart. I am therefore exploring the use of histidine as an alternative. But I am noticing several weird behaviours that I am hoping someone can shine some light on. Most alarmingly, the histidine appears to strip the nickel from the column. At the end the resin is pure white, almost as if I had run EDTA over it. Can anyone please explain why this might happen? This stripping might also account why my proteins elute in just 20mM histidine, whereas it usually elutes in 200mM imidazole. Can anyone comment on the relative 'potency' of histidine-mediated elution compared to imidazole? I would have thought they were roughly equivalent. For the record, my binding buffer is 20mM Tris pH8.0, 150mM NaCl, 0.5% DM* *DM is a detergent, these proteins are membrane-bound in vivo. the technical manual says that <1% detergent is fine and presents no problem when usuing imidazole for elution. Thank you in advance Simon From engelbert_buxbaum from hotmail.com Fri May 29 12:07:27 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri May 29 17:53:35 2009 Subject: expression of a subunit References: Message-ID: Am 26.05.2009, 12:50 Uhr, schrieb Azam Rahimpour : > I am going to express a mutant subunit of a multimeric complex protein > in mammalian cells , this subunit is expected to incorporate in the > whole protein complex, I dont know if attachment of small tags like his > tag or HA tag would disrupt its structure and prevent its incorporation > in a complex. does any one has any experience? If the 3D structure has been solved (check the PDB), you can look wether C- or N-terminus is free (solvent exposed). If that is the case, puting a tag there should not overly affect folding and association. The same is true for exposed loops, but keep in mind that they may be a binding site for regulatory proteins (if that is important to you). If the structure is not known, then you'll have to use trial and error. From celesteel from gmail.com Fri May 29 18:00:04 2009 From: celesteel from gmail.com (L Celeste) Date: Fri May 29 21:03:58 2009 Subject: Help with PCR reaction Phusion Flash - wrong sized fragment In-Reply-To: <783f0aF1k93q4U1@mid.individual.net> References: <783f0aF1k93q4U1@mid.individual.net> Message-ID: <7f3233410905291600q16f10f00qbdde03a5adb8502@mail.gmail.com> Thanks everyone for your input. I will try the suggestion and see if it works! From celesteel from gmail.com Fri May 29 18:05:48 2009 From: celesteel from gmail.com (L Celeste) Date: Fri May 29 21:04:09 2009 Subject: Restriction Enzyme Help: Sac II Message-ID: <7f3233410905291605i5fd58267t2b59a83927e97295@mail.gmail.com> Hi everyone, I have been trying to amplify two segments ( 9 kb and 11 kb). I was able to amplify the right-zed fragment with a primer set containing Sac II. After restriction enzyme digestion with Sac II, I tried to ligate these two fragments together. But they fail to ligate but the positve control worked. Any help would be much appreciated. Thank you! Celeste From nobody from nospam.not Fri May 29 20:10:19 2009 From: nobody from nospam.not (Han) Date: Fri May 29 21:04:19 2009 Subject: no antibiotics when transfecting/infecting. Why? Message-ID: Just showing my ignorance. I just overheard people talk about failing infections (lentivirus/AAV2) or transfections when the cells to be infected or transfected are in antibiotic containing media. Why is this so? Thanks in advance for educating me. -- Best regards Han email address is invalid From nobody from nospam.not Fri May 29 20:25:58 2009 From: nobody from nospam.not (Han) Date: Fri May 29 21:04:26 2009 Subject: no antibiotics when transfecting/infecting. Why? References: Message-ID: dk@no.email.thankstospam.net (DK) wrote in news:BP%Tl.81347$uD3.48715@newsfe20.iad: > In article , Han > wrote: >>Just showing my ignorance. I just overheard people talk about failing >>infections (lentivirus/AAV2) or transfections when the cells to be >>infected or transfected are in antibiotic containing media. Why is >>this so? > > Details? What antybiotics - the usual anti-bacterial ones or the > selective drug? No experience with lentivirus but can tell about > transfections: if the former, then the claims are simply not true - > transfections work fine with the pen/strep or gentamicin; if the > latter, then the reason for not working should be obvious. > > DK > It was the usual pen/strep. Thanks for the quick reply. I'll have to needle my colleagues. They stated that it was well-known it didn't work, but couldn't explain why. -- Best regards Han email address is invalid From novalidaddress from nurfuerspam.de Sat May 30 05:12:54 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Sat May 30 13:29:43 2009 Subject: Restriction Enzyme Help: Sac II References: Message-ID: <5862ef37-dd04-4d02-b7a2-5dde45d87926@v4g2000vba.googlegroups.com> Hi Celeste, suppose you have removed Taq with a PCR cleanup. if you check NEB's information on Sac II (http://www.neb.com/nebecomm/ products/productR0157.asp), you'll find 2 possible explanations: 1. Certain SacII sites (e.g. one in the right arm of ? DNA) are resistant to cleavage. The reason particular SacII sites in ? DNA and ?X174 DNA are cleaved at significantly lower rates than those found with other substrates is unclear at present. 2. SacII needs to interact with two copies of its recognition sequence to cleave. As a result, substrates with single SacII sites are cleaved at a reduced rate. To overcome this, you might consider to increase enzyme concentration, DNA concentration, and digestion time. Have you enough overhang for SacII "to bite"? Good luck! Wo On May 30, 1:05?am, L Celeste wrote: > Hi everyone, > > I have been trying to amplify two segments ( 9 kb and 11 kb). I was able to > amplify the right-zed fragment with a primer set containing Sac II. After > restriction enzyme digestion with Sac II, I tried to ligate these two > fragments together. But they fail to ligate but the positve control worked. > Any help would be much appreciated. Thank you! > > Celeste From aawara from pontiff-playground.org Sat May 30 09:52:11 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Sat May 30 13:29:50 2009 Subject: no antibiotics when transfecting/infecting. Why? References: Message-ID: In , Han wrote: >> Details? What antybiotics - the usual anti-bacterial ones or the >> selective drug? No experience with lentivirus but can tell about >> transfections: if the former, then the claims are simply not true - >> transfections work fine with the pen/strep or gentamicin; if the >> latter, then the reason for not working should be obvious. >> >> DK >> > It was the usual pen/strep. Thanks for the quick reply. I'll have to > needle my colleagues. They stated that it was well-known it didn't work, > but couldn't explain why. > Agree 100% with Dima. We do lentiviral/retroviral infections all the time, in media containing antibiotics. Now anti-fungals are another matter. Many of them impact the infectivity of enveloped viruses that contain cholesterol rich lipid membranes - like retroviruses and lentiviruses. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From celesteel from gmail.com Sat May 30 19:08:31 2009 From: celesteel from gmail.com (L Celeste) Date: Sun May 31 13:32:20 2009 Subject: Restriction Enzyme Help: Sac II In-Reply-To: <5862ef37-dd04-4d02-b7a2-5dde45d87926@v4g2000vba.googlegroups.com> References: <5862ef37-dd04-4d02-b7a2-5dde45d87926@v4g2000vba.googlegroups.com> Message-ID: <7f3233410905301708j1cb1566ajf354854f2e6510e6@mail.gmail.com> Hi Wo, Thank you for your info. How do I know if my DNA has SacII sites resistant to cleavage? Also, I am not sure what you meant by 'Have you enough overhang for SacII "to bite"?' Would you mind explaining that? Thanks! Celeste 2009/5/30 WS > Hi Celeste, > > suppose you have removed Taq with a PCR cleanup. > > if you check NEB's information on Sac II (http://www.neb.com/nebecomm/ > products/productR0157.asp), > you'll find 2 possible explanations: > > 1. Certain SacII sites (e.g. one in the right arm of ? DNA) are > resistant to cleavage. The reason particular SacII sites in ? DNA and > ?X174 DNA are cleaved at significantly lower rates than those found > with other substrates is unclear at present. > 2. SacII needs to interact with two copies of its recognition > sequence to cleave. As a result, substrates with single SacII sites > are cleaved at a reduced rate. > > To overcome this, you might consider to increase enzyme concentration, > DNA concentration, and digestion time. > > Have you enough overhang for SacII "to bite"? > > Good luck! > > Wo > > On May 30, 1:05 am, L Celeste wrote: > > Hi everyone, > > > > I have been trying to amplify two segments ( 9 kb and 11 kb). I was able > to > > amplify the right-zed fragment with a primer set containing Sac II. After > > restriction enzyme digestion with Sac II, I tried to ligate these two > > fragments together. But they fail to ligate but the positve control > worked. > > Any help would be much appreciated. Thank you! > > > > Celeste > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From gerchman from research.haifa.ac.il Sun May 31 07:29:54 2009 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Sun May 31 13:32:28 2009 Subject: EDTA solution becomes slurry Message-ID: <1243772994.4a22784249cf1@webmail.haifa.ac.il> EDTA will not dissolve until pH 8 is reached, hence the "white slurry". Simply keep adding NaOH while measuring pH (I suggest using a pH paper, not an electrode). When you get near pH=8 you will see sudden clearance of the solution. You can definitely start with NaOH pellet (add a little and let it dissolve completely before adding more) and use NaOH solution only for final adjustment. Preparing EDTA solution is a slow process... Yoram ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From nobody from nospam.not Sat May 30 16:54:23 2009 From: nobody from nospam.not (Han) Date: Sun May 31 13:32:36 2009 Subject: no antibiotics when transfecting/infecting. Why? References: Message-ID: Aawara Chowdhury wrote in news:vKbUl.117450$HZ1.10056@newsfe15.iad: > In , > Han wrote: > >>> Details? What antybiotics - the usual anti-bacterial ones or the >>> selective drug? No experience with lentivirus but can tell about >>> transfections: if the former, then the claims are simply not true - >>> transfections work fine with the pen/strep or gentamicin; if the >>> latter, then the reason for not working should be obvious. >>> >>> DK >>> >> It was the usual pen/strep. Thanks for the quick reply. I'll have >> to needle my colleagues. They stated that it was well-known it >> didn't work, but couldn't explain why. >> > > Agree 100% with Dima. We do lentiviral/retroviral infections all the > time, in media containing antibiotics. Now anti-fungals are another > matter. Many of them impact the infectivity of enveloped viruses that > contain cholesterol rich lipid membranes - like retroviruses and > lentiviruses. > > AC > Thanks, Aawara. I can easily see antifungals interfering with membrane trafficking. -- Best regards Han email address is invalid From novalidaddress from nurfuerspam.de Sun May 31 15:05:03 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Sun May 31 20:31:45 2009 Subject: Restriction Enzyme Help: Sac II References: <5862ef37-dd04-4d02-b7a2-5dde45d87926@v4g2000vba.googlegroups.com> Message-ID: <5a055015-208e-4057-a7af-07a72e226e29@u10g2000vbd.googlegroups.com> Hi Celeste, if your fragments won't link, then something must be wrong with them, i.e. they do not follow your theory. One reason might be the enzyme has not cut (properly). Some enzymes only can bind (and thus cut) to DNA when there is some overhang. The NEB catalogue provides a nice chart where this effect has been tested for with several enzymes. For myself, as oligo are cheap, I generally add 4 or 5 bases extra (usually A's or T's) after an RE site which is introduced by primers to avoid any hassle because of this. Sometimes enzymes simply won't cut for unknown reasons: The recognition sequence seems to be a necessary criterion but there might be other features like secondary structure which from time to time turn engineering into science and RE-search. In that case you'll be stuck. You might try to add some DMSO, Betain, heat to your digest, as one would do with noncompliant PCRs. To find out if your PCR products have been cut well is quite difficult, as they are quite long. If there is another RE site nearby allowing to cut off a small fragment off the end of your PCR product, you could check if the size of this small fragment changes after SacII treatment. Use PAGE or capillary electrophoresis for this. This might also tell you if you get partial digestion (i.e. if incubation time / amount of enzyme / DNA concentration should be increased). You also might label the ends with radioactive phosphate or a labeled base (eg biotinylated dATP and Taq) and check if the label goes off by adding SacII. But in the end there is a big chance that you'll just know why it doesn't work and it won't get you much closer to a solution. In terms of economy, the best solution might be making new primers and use another enzyme, as another possible problem with SacII is that the overhang is only 2 bases which is generally considered to be difficult for ligation. If you need to design sort of unique RE site, SfiI is a nice tool. If you want to try again with SacII, also make sure that you add PEG to your ligation, ligate at low temperature (i.e. in the fridge oder set a cycler to 4-10 degC cycles). Also run the ligation quite dilute in order to disfavor cyclisation if you have 2 SacII sites per molecule). Good luck! Wo From tk from shaggy.csail.mit.edu Sun May 31 16:41:08 2009 From: tk from shaggy.csail.mit.edu (Tom Knight) Date: Sun May 31 20:31:53 2009 Subject: EDTA solution becomes slurry References: Message-ID: Yoram Gerchman writes: > EDTA will not dissolve until pH 8 is reached, hence the "white > slurry". Simply keep adding NaOH while measuring pH (I suggest using > a pH paper, not an electrode). When you get near pH=8 you will see > sudden clearance of the solution. You can definitely start with NaOH > pellet (add a little and let it dissolve completely before adding > more) and use NaOH solution only for final adjustment. Preparing > EDTA solution is a slow process... To save some time and frustration, you need about equimolar amounts of NaOH and EDTA to dissolve the EDTA. Effectively, you are making trisodium EDTA from disodium EDTA. You can add almost enough pelleted NaOH to reach this point, then titrate with pH paper and 5M NaOH solution to reach pH 8.0. Just a reminder, you cannot make a 1 M solution -- the stock solution is 500 mM.