From sanjiban.c from gmail.com Tue Sep 2 12:31:05 2008 From: sanjiban.c from gmail.com (sanjiban chakrabarty) Date: Tue Sep 2 17:32:11 2008 Subject: BAC Protocol Message-ID: <8403d0360809021031v5e90fe2fn6bdfe5ac418584bf@mail.gmail.com> Dear Sir, I am working with BAC DNA and I have faced lot of problem with BAC minipreps as the yield is always very low and the BAC DNA is contaminated with E Coli genomic DNA. I will be grateful if you can suggest me some protocol for the same. Waiting for your reply. Regards, Sanjiban From rory.obrien from stonebow.otago.ac.nz Tue Sep 2 18:21:45 2008 From: rory.obrien from stonebow.otago.ac.nz (Rory O'Brien) Date: Tue Sep 2 18:36:56 2008 Subject: Larger numbers of DNA extractions... Message-ID: Can anyone suggest an appropriate methodology for extracting DNA from a large number (1,000ish) of stored buffy coats? Maybe somebody has a 96 well plate DNA extraction protocol they might care to share; the commercial ones are sooo expensive! Downstream application is a simple PCR allelic discrimination assay. Many thanks, - Rory From zhiqi2 from illinois.edu Tue Sep 2 18:55:48 2008 From: zhiqi2 from illinois.edu (Zhi Qi) Date: Tue Sep 2 18:37:05 2008 Subject: A question about DNA Ligation Message-ID: <48BDD284.90102@illinois.edu> Dear all, I want to ligate a ssDNA with a ~2-kbp dsDNA with a 15-nt overhang. The ssDNA oligo has a 60-bp hairpin construct with a 15-nt overhang at 5' end. These two 15-nt overhang on ssDNA and long dsDNA are designed to complimentary to each other. I added ssDNA and dsDNA together with enough T4 DNA ligase (NEB), and ssDNA:dsDNA = 100:1 (I just hope ligation efficiency is high). I incubated the sample at 16 C for 12 hour and then 65 C for 20 min to inactive T4 ligase. From the 1% agarose gel, I can get ~2.1-kbp band which means the ssDNA + dsDNA. However, when I used gel extraction kit (QIAGEN) to cut the band out and purify it again, and run another gel to check it, this ~2.1-kbp band lost. I guess ~2.1-kbp band I got just an annealing product (15-nt overhang is long enough to anneal each other, right?), but the ligation did not work. It is because my dsDNA is too long (~2-kbp) to block the T4 ligase to bind on the ligase position? Another thing I know is that the yield of single strand ligation is lower than the normal ligation (two strand ligation) I will be grateful if you can give me some idea and suggestion. Waiting for your reply. Best Regards, Zhi Qi Biophysics Department UIUC From glenn.dunshea from gmail.com Tue Sep 2 19:14:28 2008 From: glenn.dunshea from gmail.com (Glenn Dunshea) Date: Wed Sep 3 09:02:01 2008 Subject: Larger numbers of DNA extractions... In-Reply-To: References: Message-ID: <8acca5110809021714t246cb5c3t4b220b6fb44b25b5@mail.gmail.com> There are various 96 well format cheap extraction methods that have been published in the last couple of years in molecular ecology notes. For example: A high-throughput protocol for extracting high-purity genomic DNA from plants and animals (2008) R. WHITLOCK, H. HIPPERSON, M. MANNARELLI and T. BURKE See also Elphinstone et al. Same journal a year or two before Cheers, Glenn On Wed, Sep 3, 2008 at 9:21 AM, Rory O'Brien < rory.obrien@stonebow.otago.ac.nz> wrote: > Can anyone suggest an appropriate methodology for extracting DNA from a > large number (1,000ish) of stored buffy coats? > > Maybe somebody has a 96 well plate DNA extraction protocol they might care > to share; the commercial ones are sooo expensive! > > Downstream application is a simple PCR allelic discrimination assay. > > Many thanks, > > - Rory > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From pjie2 from cam.ac.uk Wed Sep 3 03:39:06 2008 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Wed Sep 3 09:02:19 2008 Subject: A question about DNA Ligation In-Reply-To: References: Message-ID: <6i70q1Fotnm1U1@mid.individual.net> Have you checked that the ssDNA and dsDNA segments have phosphates at the appropriate ends? If you don't have the phosphates in the right places, you won't get any ligation. Peter Zhi Qi wrote: > Dear all, > > I want to ligate a ssDNA with a ~2-kbp dsDNA with a 15-nt overhang. The > ssDNA oligo has a 60-bp hairpin construct with a 15-nt overhang at 5' > end. These two 15-nt overhang on ssDNA and long dsDNA are designed to > complimentary to each other. > > I added ssDNA and dsDNA together with enough T4 DNA ligase (NEB), and > ssDNA:dsDNA = 100:1 (I just hope ligation efficiency is high). I > incubated the sample at 16 C for 12 hour and then 65 C for 20 min to > inactive T4 ligase. > > From the 1% agarose gel, I can get ~2.1-kbp band which means the ssDNA > + dsDNA. However, when I used gel extraction kit (QIAGEN) to cut the > band out and purify it again, and run another gel to check it, this > ~2.1-kbp band lost. I guess ~2.1-kbp band I got just an annealing > product (15-nt overhang is long enough to anneal each other, right?), > but the ligation did not work. It is because my dsDNA is too long > (~2-kbp) to block the T4 ligase to bind on the ligase position? > > Another thing I know is that the yield of single strand ligation is > lower than the normal ligation (two strand ligation) > > I will be grateful if you can give me some idea and suggestion. > > Waiting for your reply. > > Best Regards, > Zhi Qi > Biophysics Department > UIUC > From prae from gmx.net Wed Sep 3 05:21:53 2008 From: prae from gmx.net (Christian Praetorius) Date: Wed Sep 3 09:02:23 2008 Subject: BAC Protocol References: Message-ID: <6i76q3Fpe2aeU1@mid.individual.net> "sanjiban chakrabarty" wrote: >I am working with BAC DNA and I have faced lot of problem with BAC minipreps >as the yield is always very low and the BAC DNA is contaminated with E Coli >genomic DNA. We have either been using a modified protocol for the Qiagen Midi prep Kit (http://www1.qiagen.com/literature/render.aspx?id=450&tp=9) or the PhasePrep BAC DNA Kit from Sigma (#NA0100). Yields were usually quite low, but since we prepared the BACs only to transfer them between different bacterial strains, this was ok for us. Christian -- X-no-Sig: yes From mail.hatti from gmx.net Wed Sep 3 08:14:30 2008 From: mail.hatti from gmx.net (Hannes Schleifer) Date: Wed Sep 3 09:02:27 2008 Subject: Promotor activity studies Message-ID: <48be8db6$0$11868$3b214f66@aconews.univie.ac.at> Hi to all! I hope I found the right group for my question/request and that I am not harming any rules in here. I am looking for a reporter-vector for promotor-activity studies in mammalian cells and thought of using blue-white-screening (our cells are adherent and screening should be no problem). So therefore I am looking for a promotorless vector witht the LacZ-gene after a MCS (and poly-A-signal, ... everthing which is necessary to get expression in mammalian cells) to clone our promotor into. I searched the web and literature and found a commercially available kit from Clontech (Luminescent ?-galactosidase Reporter System 3) - does anyone of you have experiences with it (and perhaps send me a little aliquot of the two vectors p?gal-Basic & p?gal-Control provided in it). Thanks in advance and kind regards, Hannes From rijutak from googlemail.com Thu Sep 4 02:38:51 2008 From: rijutak from googlemail.com (Rijuta Kotenkar) Date: Thu Sep 4 10:35:16 2008 Subject: A question about DNA Ligation In-Reply-To: <200809031704.m83H4MU26270@net.bio.net> References: <200809031704.m83H4MU26270@net.bio.net> Message-ID: <48BF908B.1020402@gmail.com> Dear Zhi Qi, I agree with Peter. You must check whether the ssDNA and dsDNA are phosphorylated. We also had extremely low ligation product, but when we re-phosphorylated the fragments to be ligated using polynucleotide kinase, we got quite good ligation products. Rijuta > > Have you checked that the ssDNA and dsDNA segments have phosphates at > the appropriate ends? If you don't have the phosphates in the right > places, you won't get any ligation. > > Peter > > > > Zhi Qi wrote: > >> Dear all, >> >> I want to ligate a ssDNA with a ~2-kbp dsDNA with a 15-nt overhang. The >> ssDNA oligo has a 60-bp hairpin construct with a 15-nt overhang at 5' >> end. These two 15-nt overhang on ssDNA and long dsDNA are designed to >> complimentary to each other. >> >> I added ssDNA and dsDNA together with enough T4 DNA ligase (NEB), and >> ssDNA:dsDNA = 100:1 (I just hope ligation efficiency is high). I >> incubated the sample at 16 C for 12 hour and then 65 C for 20 min to >> inactive T4 ligase. >> >> From the 1% agarose gel, I can get ~2.1-kbp band which means the ssDNA >> + dsDNA. However, when I used gel extraction kit (QIAGEN) to cut the >> band out and purify it again, and run another gel to check it, this >> ~2.1-kbp band lost. I guess ~2.1-kbp band I got just an annealing >> product (15-nt overhang is long enough to anneal each other, right?), >> but the ligation did not work. It is because my dsDNA is too long >> (~2-kbp) to block the T4 ligase to bind on the ligase position? >> >> Another thing I know is that the yield of single strand ligation is >> lower than the normal ligation (two strand ligation) >> >> I will be grateful if you can give me some idea and suggestion. >> >> Waiting for your reply. >> >> Best Regards, >> Zhi Qi >> Biophysics Department >> UIUC >> >> > > > ------------------------------ > > Message: 6 > Date: Wed, 03 Sep 2008 10:21:53 +0000 > From: Christian Praetorius > Subject: Re: BAC Protocol > To: methods@net.bio.net > Message-ID: <6i76q3Fpe2aeU1@mid.individual.net> > Content-Type: text/plain; charset=us-ascii > > "sanjiban chakrabarty" wrote: > > >> I am working with BAC DNA and I have faced lot of problem with BAC minipreps >> as the yield is always very low and the BAC DNA is contaminated with E Coli >> genomic DNA. >> > > We have either been using a modified protocol for the Qiagen Midi prep > Kit (http://www1.qiagen.com/literature/render.aspx?id=450&tp=9) or the > PhasePrep BAC DNA Kit from Sigma (#NA0100). Yields were usually quite > low, but since we prepared the BACs only to transfer them between > different bacterial strains, this was ok for us. > > Christian > > From Paul.Phelan from tufts.edu Thu Sep 4 22:25:47 2008 From: Paul.Phelan from tufts.edu (Paul J. Phelan) Date: Fri Sep 5 10:31:55 2008 Subject: pET15b (Novagen) IPTG induction Message-ID: <20080904232547.sk1wcgejuo04gk0s@webmail.tufts.edu> I am having trouble with inducing expression of a protein that I have cloned into pET15b (Novagen). I have tried induction in BL21 E. coli with 1.0 mM IPTG in LB/100 ug/ml Ampicillin, at an OD(600 nm) of 0.5-0.6, at 37 C for up to 8 hrs and then overnight. Harvested cells are lysed by 30 min. incubation with 1 mg/ml lysozyme, followed by sonication (6x 10s for small scale 4 ml lysates, prepared from up to 100 ml cultures). But so far, my IPTG-induced lysates on SDS-PAGE look just the same as a non-induced control; I see no IPTG-dependent band at 52 kDa where I want to see one. I have re-sequenced my plasmid after transformation into BL21, and the sequence is correct. Am I missing something somewhere? Any enlightenment would be greatly appreciated Paul Phelan Tufts University Department of Biochemistry Boston From m.d.jones from imperial.ac.uk Fri Sep 5 07:03:19 2008 From: m.d.jones from imperial.ac.uk (Michael David Jones) Date: Fri Sep 5 10:32:08 2008 Subject: E. coli Strain Request In-Reply-To: <200809031704.m83H4MU26270@net.bio.net> Message-ID: Hi We wish to use lactose (and not IPTG) to induce expression of genes cloned into pGEX vectors (under control of the lac operator) for large scale protein purification. To utilise lactose, as opposed to IPTG, requires an E. coli strain that has a wild type lac operon, wild type gal operon and ideally ompT and lon defective to prevent protease degradation of the expressed gene. Anyone out there have such an E.coli strain or suggestions? Most lab strains appear to be mutated in the lac and/or gal operons. Mick Jones From holeung from berkeley.edu Fri Sep 5 13:34:51 2008 From: holeung from berkeley.edu (Ho-Leung Ng) Date: Fri Sep 5 22:14:48 2008 Subject: pET15b (Novagen) IPTG induction Message-ID: <6678762a0809051134t12884bc3yc0c1aca5ad7704ab@mail.gmail.com> Some constructs just won't express in E. coli, especially if it's eukaryotic in origin. My first suggestion is to try the expression at room temperature. Otherwise, change the construct, such as adding a fusion (His, MBP, GST, etc.) tag. Another possibility is that your protein is toxic. You can check by seeing if there is cell lysis or poor growth. ho ----------------------------------------------------------------------------------------------- I am having trouble with inducing expression of a protein that I have cloned into pET15b (Novagen). I have tried induction in BL21 E. coli with 1.0 mM IPTG in LB/100 ug/ml Ampicillin, at an OD(600 nm) of 0.5-0.6, at 37 C for up to 8 hrs and then overnight. Harvested cells are lysed by 30 min. incubation with 1 mg/ml lysozyme, followed by sonication (6x 10s for small scale 4 ml lysates, prepared from up to 100 ml cultures). But so far, my IPTG-induced lysates on SDS-PAGE look just the same as a non-induced control; I see no IPTG-dependent band at 52 kDa where I want to see one. I have re-sequenced my plasmid after transformation into BL21, and the sequence is correct. Am I missing something somewhere? Any enlightenment would be greatly appreciated Paul Phelan Tufts University Department of Biochemistry Boston From bmacgreg from unc.edu Fri Sep 5 14:00:28 2008 From: bmacgreg from unc.edu (Barbara MacGregor) Date: Fri Sep 5 22:14:56 2008 Subject: E. coli strain request In-Reply-To: <200809051704.m85H4OV21058@net.bio.net> References: <200809051704.m85H4OV21058@net.bio.net> Message-ID: Hello, I can't recommend a particular strain, but you can search for strains by genotype at CGSC: http://cgsc2.biology.yale.edu/index.php Sorry if you've already looked there! Barbara MacGregor > > > We wish to use lactose (and not IPTG) to induce expression of genes > cloned > into pGEX vectors (under control of the lac operator) for large scale > protein purification. To utilise lactose, as opposed to IPTG, > requires an > E. coli strain that has a wild type lac operon, wild type gal operon > and > ideally ompT and lon defective to prevent protease degradation of the > expressed gene. > > Anyone out there have such an E.coli strain or suggestions? Most lab > strains appear to be mutated in the lac and/or gal operons. > > Mick Jones From engelbert_buxbaum from hotmail.com Fri Sep 5 14:11:33 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri Sep 5 22:15:02 2008 Subject: Counterstain for AEC AND BCIP/NBT References: Message-ID: Am 28.08.2008, 03:06 Uhr, schrieb Bluescript : > On 27 Aug., 21:10, "Dr Engelbert Buxbaum" > wrote: >> Am 27.08.2008, 04:30 Uhr, schrieb Bluescript : >> >> > Hi, >> > I want to do immunohistochemistry detecting two targets at the same >> > time. For various reasons I am going to use AEC as substrate for HRP >> > and BCIP/NBT as AP substrate. Now I am looking for a good >> > counterstain. Because of the AEC the counterstain has to work with an >> > aqueous embedding medium. > Thank you very much for your suggestions. Maybe I forgot to mention > that I am looking for a chromogenic counterstain that can be > visualized by light microscopy! Classical nuclear stains like haematoxilin, methylgreen, nuclear fast red should work, perhaps you'll have to apply them before immunostaining. Methylenblue-eosinate (Giemsa, Wright, Papenheim...) would give blue nuclei and pink cytosol, you'll have to check whether the contrast with the immunostaining is sufficient. Perhaps switch to Diaminobenzidine/Nickel for HRP detection (black, chemically quite inert). But overall, I'd recommend swithching to fluorescence detection, check http://probes.invitrogen.com/handbook/ From elz.lis from gmail.com Mon Sep 8 06:00:24 2008 From: elz.lis from gmail.com (E. Lis) Date: Mon Sep 8 10:29:43 2008 Subject: pET15b (Novagen) IPTG induction Message-ID: <48C505C8.4@gmail.com> It is possible that your protein is present in inclusion bodies, and lysozyme and sonication will not disrupt those. Have you tried lysis by 8M urea? You could also try running entire bacterial cells suspended in SDS-PAGE sample buffer (as 10 min in sample buffer at 100^C should do the trick). From scoop from mail.nih.gov Wed Sep 10 00:26:55 2008 From: scoop from mail.nih.gov (Sharon Cooperman) Date: Wed Sep 10 08:34:26 2008 Subject: problem with degradation of cytoplasmic RNA fraction when making cytoplasmic and nuclear fractionated RNA Message-ID: Hi, I am trying to make fractionated cytoplasmic and nuclear RNA from mouse bone marrow and MEL (erythroleukemia) cells. I have tried the Qiagen alternate protocol (on their web site) and the Norgen Biotek nuclear/cytoplasmic RNA fractionation kit. My nuclear RNA looks great, but my cytoplasmic RNA looks very degraded (no ribosomal bands). I know that there is little protection from RNase during the cytoplasmic lysis/nuclear spin down steps of the protocol and I wonder if I'm getting degradation of my cytoplasmic RNA during those steps of the protocol. On the Norgen web site they show beautiful photos of undegraded ribosomal bands of cytoplasmic RNA made using their kit, but they are using HeLa cells and I wonder if bone marrow has more RNase than HeLa cells and that's why my result is not as good. Does anyone have any experience isolating cytoplasmic RNA and any suggestions for me (such as a better protocol, a way to inhibit RNase, what RNase inhibitors to use, etc)? Any advice or encouragement will be greatly appreciated! Thanks! Sharon -- Sharon Cooperman NIH, NICHD, CBMB 301.741-6092 Building 18T, room 101 301.402-0078 fax Bethesda, MD 20892 From anirbn from gmail.com Sat Sep 13 00:46:25 2008 From: anirbn from gmail.com (ab) Date: Sat Sep 13 08:21:15 2008 Subject: Sequence alignment from BLAST output Message-ID: <111da47d-b7f3-4f87-9bb3-823b8fb0fa9e@c58g2000hsc.googlegroups.com> Dear all, Sorry about the silly question. Is there any website where one can BLAST microbial genomes with a protein sequence, select a subset from the BLAST output and make a multiple sequence alignment (with CLUSTAL_W or equivalent) without the need to individually copy/paste every selected sequence into a separate file ? Thanks very much in advance. Best, Anirban. From R.Jayakumar from roswellpark.org Sat Sep 13 14:59:09 2008 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Sat Sep 13 19:46:30 2008 Subject: Sequence alignment from BLAST output In-Reply-To: <111da47d-b7f3-4f87-9bb3-823b8fb0fa9e@c58g2000hsc.googlegroups.com> References: <111da47d-b7f3-4f87-9bb3-823b8fb0fa9e@c58g2000hsc.googlegroups.com> Message-ID: <97101976F8A044468CA74FE11883B90E173E7B16@VISTA.roswellpark.org> If there is, you should find a link from www.expasy.ch or www.bioinformatik.de, two of the largest repositories of molecular tools on the web. But I wish there were tools which will do all our work while we relax with a martini in front of the fireplace, but unfortunately, some amount of work is required. :-)) Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of ab Sent: Saturday, September 13, 2008 1:46 AM To: methods@magpie.bio.indiana.edu Subject: Sequence alignment from BLAST output Dear all, Sorry about the silly question. Is there any website where one can BLAST microbial genomes with a protein sequence, select a subset from the BLAST output and make a multiple sequence alignment (with CLUSTAL_W or equivalent) without the need to individually copy/paste every selected sequence into a separate file ? Thanks very much in advance. Best, Anirban. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. From dprieto from fcien.edu.uy Mon Sep 15 10:42:43 2008 From: dprieto from fcien.edu.uy (Daniel Prieto) Date: Mon Sep 15 11:38:53 2008 Subject: Plasmid elution from filter problem Message-ID: <48CE8273.40206@fcien.edu.uy> Hi all, I am trying to elute some plasmids from a whatman paper but do not seem to achieve it. I tried the classic 50 uL water, 5 minutes at RT and spinned down the paper. I am usually quite successful using this technique with other plasmids. I "eluted" them this way and tried to transform (2 different methods) with no results, I ran the DNA on an agarose gel but could not see any band. The guys who sent it to me told me they were concentrated enough to see them quite well with EtBr. I have had them for almost a year in the filter prior to my attempts to elute them, but I don't see much of a problem in that. Can someone give me some suggestions to improve my yield with the elution? Thank you, Daniel From mrnance from lsi.umich.edu Mon Sep 15 14:37:27 2008 From: mrnance from lsi.umich.edu (Mark Nance) Date: Mon Sep 15 17:43:08 2008 Subject: Sequence alignment from BLAST output Message-ID: <48CE8138020000B50000DD9A@lsigroupwise01.lsi.umich.edu> Anirban, You can use the protein blast from pubmed. After your blast, scroll down and check the boxes next to the sequences you want. Click the "Get Selected Sequences" box. On the Results page it sends you to, pull down the Display option to get FASTA. Pull down the Send To option, and choose text. You can then copy/paste all of the sequences you selected into ClustalW. You still have to copy and paste, but you only have to do it once. Mark >>> 09/14/08 1:04 PM >>> 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: Sequence alignment from BLAST output (Jayakumar, R) ---------------------------------------------------------------------- Message: 1 Date: Sat, 13 Sep 2008 15:59:09 -0400 From: "Jayakumar, R" Subject: RE: Sequence alignment from BLAST output To: "ab" , Message-ID: <97101976F8A044468CA74FE11883B90E173E7B16@VISTA.roswellpark.org> Content-Type: text/plain; charset="us-ascii" If there is, you should find a link from www.expasy.ch or www.bioinformatik.de, two of the largest repositories of molecular tools on the web. But I wish there were tools which will do all our work while we relax with a martini in front of the fireplace, but unfortunately, some amount of work is required. :-)) Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of ab Sent: Saturday, September 13, 2008 1:46 AM To: methods@magpie.bio.indiana.edu Subject: Sequence alignment from BLAST output Dear all, Sorry about the silly question. Is there any website where one can BLAST microbial genomes with a protein sequence, select a subset from the BLAST output and make a multiple sequence alignment (with CLUSTAL_W or equivalent) without the need to individually copy/paste every selected sequence into a separate file ? Thanks very much in advance. Best, Anirban. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 40, Issue 9 ************************************** !DSPAM:63,48cd4473197321173833723! From mron003 from hotmail.com Mon Sep 15 19:40:41 2008 From: mron003 from hotmail.com (Miguel _) Date: Mon Sep 15 22:30:52 2008 Subject: locus info Message-ID: Hi, I'm trying to relate the AffyID to the genomic position and locus name for that spot. I'm using ygs98 chips. I'm new to Bioconductor and R and I'm not sure how to do this. Cheers, Miguel _________________________________________________________________ Find singles in your area with Match. http://a.ninemsn.com.au/b.aspx?URL=http%3A%2F%2Fmatch%2Enz%2Emsn%2Ecom%2Fchannel%2Findex%2Easpx%3Ftrackingid%3D1043416&_r=WL_EMAL_TAG&_m=EXT From itisam.sarangi from gmail.com Tue Sep 16 10:27:34 2008 From: itisam.sarangi from gmail.com (Itisam Sarangi) Date: Tue Sep 16 16:12:36 2008 Subject: info abt linker in GFP fusion protein Message-ID: <25de2fb70809160827j192229cfx6e6b6bc6e90bbdef@mail.gmail.com> Hi all, I am new to the cell biology field. I am presently trying to tag eGFP to my protein of intrest in Pmyc vector. I am trying to prepare a vector with MCS before and after eGFP. what shd be the minimum length of linker so tht I can see GFP in cell after transfection? which one is better C -terminal or N terminal tagged? What if I give MCS before or after GFP so tht gfp remains in frame still I can put any other protein? where can I find regarding MCS any help in this matter will be helpful. Thanking you -- Itisam Sarangi From chen219 from purdue.edu Tue Sep 16 20:12:29 2008 From: chen219 from purdue.edu (chen219@purdue.edu) Date: Wed Sep 17 12:28:56 2008 Subject: Why is NPM localized in nucleoplasm in drug treatment control cells? Message-ID: <1221613949.48d0597d3a484@webmail.purdue.edu> I treated A549 and Hela cells with ActD, MPA, DOX and Anisomycin, using DMSO, EtOH, H2O and MeOH as vehicle control, respectively. NPM is localized in nucleoplasm in all the cells including vehicle control cells. I don't know how to explain this phenomenon. Have anyone had any experience in this kind of experiments? Does anyone have any thoughts? Li From anirbn from gmail.com Wed Sep 17 00:41:42 2008 From: anirbn from gmail.com (ab) Date: Wed Sep 17 12:29:02 2008 Subject: Sequence alignment from BLAST output References: Message-ID: <36cee3da-3b65-424d-8fd5-5377604a9f7f@p10g2000prf.googlegroups.com> Thanks a ton Mark. That was quite helpful. Best, Anirban. On Sep 15, 3:37?pm, "Mark Nance" wrote: > Anirban, > > You can use the protein blast from pubmed. ?After your blast, scroll down and check the boxes next to the sequences you want. ?Click the "Get Selected Sequences" box. ?On the Results page it sends you to, pull down the Display option to get FASTA. ?Pull down the Send To option, and choose text. ?You can then copy/paste all of the sequences you selected into ClustalW. ?You still have to copy and paste, but you only have to do it once. > > Mark > > >>> 09/14/08 1:04 PM >>> > > Send Methods mailing list submissions to > ? ? ? ? meth...@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-requ...@net.bio.net > > You can reach the person managing the list at > ? ? ? ? methods-ow...@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: Sequence alignment from BLAST output (Jayakumar, R) > > ---------------------------------------------------------------------- > > Message: 1 > Date: Sat, 13 Sep 2008 15:59:09 -0400 > From: "Jayakumar, R" > Subject: RE: Sequence alignment from BLAST output > To: "ab" , > Message-ID: > ? ? ? ? <97101976F8A044468CA74FE11883B90E173E7...@VISTA.roswellpark.org> > Content-Type: text/plain; charset="us-ascii" > > If there is, you should find a link fromwww.expasy.chorwww.bioinformatik.de, two of the largest repositories of molecular tools > on the web. ?But I wish there were tools which will do all our work > while we relax with a martini in front of the fireplace, but > unfortunately, some amount of work is required. ?:-)) > Jay > > -----Original Message----- > From: methods-boun...@oat.bio.indiana.edu > > [mailto:methods-boun...@oat.bio.indiana.edu] On Behalf Of ab > Sent: Saturday, September 13, 2008 1:46 AM > To: meth...@magpie.bio.indiana.edu > Subject: Sequence alignment from BLAST output > > Dear all, > > Sorry about the silly question. Is there any website where one can BLAST > microbial genomes with a protein sequence, select a subset from the > BLAST output and make a multiple sequence alignment (with CLUSTAL_W or > equivalent) without the need to individually copy/paste every selected > sequence into a separate file ? > > Thanks very much in advance. > > Best, > > Anirban. > _______________________________________________ > Methods mailing list > Meth...@net.bio.nethttp://www.bio.net/biomail/listinfo/methods > > This email message may contain legally privileged and/or confidential information. ?If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. ?If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. > > ------------------------------ > > _______________________________________________ > Methods mailing list > Meth...@net.bio.nethttp://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 40, Issue 9 > ************************************** > > !DSPAM:63,48cd4473197321173833723! From aawara from pontiff-playground.org Wed Sep 17 08:17:29 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Wed Sep 17 12:29:19 2008 Subject: Plasmid elution from filter problem References: Message-ID: In , Daniel Prieto wrote: > Hi all, I am trying to elute some plasmids from a whatman paper but do > not seem to achieve it. I tried the classic 50 uL water, 5 minutes at RT > and spinned down the paper. I am usually quite successful using this > technique with other plasmids. I "eluted" them this way and tried to > transform (2 different methods) with no results, I ran the DNA on an > agarose gel but could not see any band. The guys who sent it to me told > me they were concentrated enough to see them quite well with EtBr. I > have had them for almost a year in the filter prior to my attempts to > elute them, but I don't see much of a problem in that. Can someone give > me some suggestions to improve my yield with the elution? Are you sure they're on Whatman filter paper, and not on Whatman DE-81 paper? I was recently sent some plasmids spotted on what was assumed to be filter paper - I could not elute them with water or TE, but they came off in high salt (1M sodium acetate, pH 5.5), and I transformed them after drop-dialysis. About a week after this happened, I received an email from the colleague I had requested the plasmids from indicating that his new technician had been sending plasmids spotted on DE81 paper and not 3mm paper, and that they would be sending the plasmids again ..... AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From marisouzamari from globo.com Wed Sep 17 12:27:52 2008 From: marisouzamari from globo.com (Mariana Castanon de Souza) Date: Wed Sep 17 12:33:23 2008 Subject: UV Absorption Spectra of Nucleic Acids Message-ID: <697effaa0809171027l5e1dea73td5e929de7aaab61c@mail.gmail.com> hello, my name is mariana casta?on and I'm a student of braislian university of rio de janeiro(UFRJ) and brasilian center of physics research (CBPF). now i'm starting to work with plamidial DNA and I need to do caracterization of that. So i wan't to ask if can you send me the differences of spectromerter of differents nucleic acids. Do you have that? Thanks. The best, M. Castanon. From dprieto from fcien.edu.uy Wed Sep 17 14:40:27 2008 From: dprieto from fcien.edu.uy (Daniel Prieto) Date: Wed Sep 17 17:49:50 2008 Subject: Plasmid elution from filter problem Message-ID: <48D15D2B.3020403@fcien.edu.uy> Great info! Didn't know the type of paper was that critical. I will try the high salt elution. Thank you! Daniel Aawara Chowdhury wrote: > In , > Daniel Prieto wrote: > > >> Hi all, I am trying to elute some plasmids from a whatman paper but do >> not seem to achieve it. I tried the classic 50 uL water, 5 minutes at RT >> and spinned down the paper. I am usually quite successful using this >> technique with other plasmids. I "eluted" them this way and tried to >> transform (2 different methods) with no results, I ran the DNA on an >> agarose gel but could not see any band. The guys who sent it to me told >> me they were concentrated enough to see them quite well with EtBr. I >> have had them for almost a year in the filter prior to my attempts to >> elute them, but I don't see much of a problem in that. Can someone give >> me some suggestions to improve my yield with the elution? >> > > Are you sure they're on Whatman filter paper, and not on Whatman DE-81 > paper? I was recently sent some plasmids spotted on what was assumed > to be filter paper - I could not elute them with water or TE, but they > came off in high salt (1M sodium acetate, pH 5.5), and I transformed > them after drop-dialysis. > > About a week after this happened, I received an email from the colleague > I had requested the plasmids from indicating that his new technician > had been sending plasmids spotted on DE81 paper and not 3mm paper, and > that they would be sending the plasmids again ..... > > AC > > From pow.joshi from gmail.com Wed Sep 17 13:25:45 2008 From: pow.joshi from gmail.com (Pow Joshi) Date: Wed Sep 17 17:50:02 2008 Subject: Plasmid elution from filter problem In-Reply-To: References: Message-ID: <710764ea0809171125s63152821u484a8790130b08f5@mail.gmail.com> 2008/9/17 Aawara Chowdhury > In , > Daniel Prieto wrote: > > > Hi all, I am trying to elute some plasmids from a whatman paper but do > > not seem to achieve it. I tried the classic 50 uL water, 5 minutes at RT > > and spinned down the paper. I am usually quite successful using this > > technique with other plasmids. I "eluted" them this way and tried to > > transform (2 different methods) with no results, I ran the DNA on an > > agarose gel but could not see any band. The guys who sent it to me told > > me they were concentrated enough to see them quite well with EtBr. I > > have had them for almost a year in the filter prior to my attempts to > > elute them, but I don't see much of a problem in that. Can someone give > > me some suggestions to improve my yield with the elution? > > Are you sure they're on Whatman filter paper, and not on Whatman DE-81 > paper? I was recently sent some plasmids spotted on what was assumed > to be filter paper - I could not elute them with water or TE, but they > came off in high salt (1M sodium acetate, pH 5.5), and I transformed > them after drop-dialysis. > > About a week after this happened, I received an email from the colleague > I had requested the plasmids from indicating that his new technician > had been sending plasmids spotted on DE81 paper and not 3mm paper, and > that they would be sending the plasmids again ..... Thanks AC, this is good to know. One of my colleagues had similar trouble an year ago, and I am now wondering if we should've tried the high salt/pH way. Pow > > > AC > -- > Email: echo 36434455860060025978157675027927670979097959886449930P | dc > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From vinodasundi from gmail.com Wed Sep 17 15:55:35 2008 From: vinodasundi from gmail.com (vinodasundi@gmail.com) Date: Wed Sep 17 17:50:06 2008 Subject: pET15b (Novagen) IPTG induction References: Message-ID: On Sep 4, 8:25?pm, "Paul J. Phelan" wrote: > I am having trouble with inducing expression of a protein that I have > cloned into pET15b (Novagen). ?I have tried induction in BL21 E. coli > with 1.0 mM IPTG in LB/100 ug/ml Ampicillin, at an OD(600 nm) of > 0.5-0.6, at 37 C for up to 8 hrs and then overnight. ?Harvested cells > are lysed by 30 min. incubation with 1 mg/ml lysozyme, followed by > sonication (6x 10s for small scale 4 ml lysates, prepared from up to > 100 ml cultures). ?But so far, my IPTG-induced lysates on SDS-PAGE look > just the same as a non-induced control; I see no IPTG-dependent band at > 52 kDa where I want to see one. ?I have re-sequenced my plasmid after > transformation into BL21, and the sequence is correct. ?Am I missing > something somewhere? > > Any enlightenment would be greatly appreciated > > Paul Phelan > Tufts University > Department of Biochemistry > Boston Hi Paul, You want to use BL21(DE3) cells and not just BL21. The former cells contain a ? prophage carrying the T7 RNA polymerase gene and lacIq. When you add IPTG to the culture, it binds to the lac repressor coded by the lacIq, allowing expression of the T7 RNA ploymerase which in turn binds to the T7 promoter on the pET plasmid and turns on genes downstream of the promoter. Good Luck! From shenq from purdue.edu Thu Sep 18 08:18:57 2008 From: shenq from purdue.edu (Qingwu Shen) Date: Thu Sep 18 11:51:01 2008 Subject: problems with protein expression Message-ID: <1221743937.48d255414f9c4@webmail.purdue.edu> Hi everybody, I'm trying to expression some rabbit skeletal TnI using E.coli. The plasmid I'm using came from some other lab and has been stored in freezer for many years. I'm using Rossetta (DE3) PlysS to express protein and the yield is very low. Even worse after I changed two Cys to Ser in the protein using PCR mutagenesis, I got no protein (mutant TnI) expression although there was a induction band on SDS - PAGE gel when compared the induced sample to non-induced control. Could anybody help me to figure out this problems? I just started to express protein using E.coli. Any help is highly appreciated. Shen From nick.theodorakis from gmail.com Thu Sep 18 10:10:31 2008 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Thu Sep 18 11:51:05 2008 Subject: Plasmid elution from filter problem References: Message-ID: On Sep 17, 3:40?pm, Daniel Prieto wrote: > Great info! Didn't know the type of paper was that critical. I will try > the high salt elution. Thank you! > Daniel DE81 is not just plain paper -- it's ion-exhange paper. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From Philomina.Selvam from usz.ch Thu Sep 18 08:31:54 2008 From: Philomina.Selvam from usz.ch (Selvam-Akumulla Philomina) Date: Thu Sep 18 11:57:15 2008 Subject: Removal of Melanin from DNA/RNA Message-ID: <8FB313F32601EF49A8D97A322DC1096B04C8B2F2@DS-00180.usz.ch> Respected Sir I am Mrs.Selvam working in Dermatology Skin Cancer Research. Our major projects are Melanoma and Lymphoma. I want to try GITC to remove melanin from biopsie, later extract DNA for HLA Typing. Can you help me right recepie of GITC, I have the following recepie for GITC: 5.0M Guanidine Isothiocyanate 8% Mercap-toethanol 10mM EDTA 50mM Tris pH 7.5 weather it is right one or not. attachment of protokol. Thanks in advance With Regards Philomina Selvam From asdhandu from trevigen.com Thu Sep 18 16:59:44 2008 From: asdhandu from trevigen.com (Solomon Dhandu) Date: Thu Sep 18 21:36:36 2008 Subject: Salmon vs. Herring Testes Message-ID: Randal: I saw your post in methods regarding fridge full of herring sperm DNA from Sigma in your fridge. Do you still have it and are you planning to use it? If you have it and are not planning to use it; I am very interested in buying it from you. We are a biotech company who manufacture DNA solution following proprietary methods. Looking forward to your reply. Solomon Dhandu Production Manager 8405 Helgerman Court Gaithersburg, MD 20877 Phone: 301-216-2800 Fax: 301-560-4973 From asdhandu from trevigen.com Thu Sep 18 17:18:20 2008 From: asdhandu from trevigen.com (Solomon Dhandu) Date: Thu Sep 18 21:36:43 2008 Subject: Biotinylated NAD Message-ID: Hi, Does anybody have a protocol to bitonylate NAD? Solomon Dhandu Production Manager 8405 Helgerman Court Gaithersburg, MD 20877 Phone: 301-216-2800 Fax: 301-560-4973 From vetdrrkc from gmail.com Thu Sep 18 13:22:25 2008 From: vetdrrkc from gmail.com (Ratan K) Date: Thu Sep 18 21:36:48 2008 Subject: brdu staining and RNA isolation later Message-ID: Hi Does anyone can give me clue how to isolate RNA from the BrdU stained frozen mammary tissue section. I am looking for a short protocol for brdu staining that do not involve antigen retriecal or if do then this step should not degrade RNA quality inside the labeled cells. All kinds of ideas will be highly appreciated. Thanks in advance. -- Cheers Ratan From asdhandu from trevigen.com Thu Sep 18 17:19:35 2008 From: asdhandu from trevigen.com (Solomon Dhandu) Date: Thu Sep 18 21:36:52 2008 Subject: Herring Sperm DNA Message-ID: Hello, Does anyone know of raw material vendor for sodium salt of herring sperm dna. Sigma is on backorder and I am looking to see if there are any other vendors of this seasonal item. Thanks, Solomon Dhandu From C.P.Pena-Diaz from 2006.hull.ac.uk Fri Sep 19 04:32:25 2008 From: C.P.Pena-Diaz from 2006.hull.ac.uk (Carmen P Pena Diaz) Date: Fri Sep 19 08:29:58 2008 Subject: problems with protein expression References: <%zCAk.9198$PS3.8322@newsfe06.iad> Message-ID: <445800D8FD5D3D43878FD7D2842A3047435406@EXCL2VS2.adir.hull.ac.uk> Hi... Concerning your protein expression issue, i should add that the rossetta strain growth rate is very low compared to others. So you should induce for longer times to obtain a better yield. Also, did you check the protein through western blot? Probably it is there and the yield is so low is not detectable on an SDS-PAGE compared to the negative control. P. -----Original Message----- From: methods-bounces@oat.bio.indiana.edu on behalf of DK Sent: Fri 19/09/2008 01:42 To: methods@magpie.bio.indiana.edu Subject: Re: problems with protein expression In article , Qingwu Shen wrote: >Hi everybody, > >I'm trying to expression some rabbit skeletal TnI using E.coli. The plasmid I'm > >using came from some other lab and has been stored in freezer for many years. > I'm >using Rossetta (DE3) PlysS to express protein and the yield is very low. Even >worse after I changed two Cys to Ser in the protein using PCR mutagenesis, I > got >no protein (mutant TnI) expression although there was a induction band on SDS - >PAGE gel when compared the induced sample to non-induced control. That would say that there was expression, wouldn't it? Did you mean to say "soluble expression"? Well, some proteins don't fold well in E.coli, that's a fact of life. Troponin I is small enough and, supposedly, simple enough in its structure, that refolding might do the job nicely. Also, some proteins don't *express* well in E.coli. May be a codon usage thing or something more complicated. I.e., we've never been able to express large amounts of yeast calmodulin in E.coli - even though bovine expresses ridiculouslywell. And that is in a strain that provides all the tRNAs that are rare in E.coli. DK _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods -------------- next part -------------- ***************************************************************************************** To view the terms under which this email is distributed, please go to http://www.hull.ac.uk/legal/email_disclaimer.html ***************************************************************************************** From davidminde from gmail.com Fri Sep 19 06:32:57 2008 From: davidminde from gmail.com (David-Paul Minde) Date: Fri Sep 19 08:30:17 2008 Subject: problems with protein expression In-Reply-To: <1221743937.48d255414f9c4@webmail.purdue.edu> References: <1221743937.48d255414f9c4@webmail.purdue.edu> Message-ID: <123243900809190432odfebc97ra0d4ca4fdf9791e4@mail.gmail.com> Dear Shen, ... your protein might be constantly degraded (and therefore be "invisible" even though it is actually highly "expressed"). Try low-temperature expression (e.g. 18?C or 15 ?C[maybe w/o antibiotics]) or even 10?C if you have arcticexpress strains. best wishes, David PS ... if it's that small, you might think of simply redesigning of your gene and just order the (e.g. E. coli) codon optimized cDNA. 2008/9/18 Qingwu Shen > Hi everybody, > > I'm trying to expression some rabbit skeletal TnI using E.coli. The plasmid > I'm > using came from some other lab and has been stored in freezer for many > years. I'm > using Rossetta (DE3) PlysS to express protein and the yield is very low. > Even > worse after I changed two Cys to Ser in the protein using PCR mutagenesis, > I got > no protein (mutant TnI) expression although there was a induction band on > SDS - > PAGE gel when compared the induced sample to non-induced control. Could > anybody > help me to figure out this problems? I just started to express protein > using > E.coli. Any help is highly appreciated. > > Shen > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 mobile phone +31(0)631154267 private address: Griftkade 4 bis 3572 TW Utrecht I never think of the future. It comes soon enough. Albert Einstein Res severa verum gaudium. (~true delight is a severe issue) Seneca From aawara from pontiff-playground.org Fri Sep 19 07:39:26 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Fri Sep 19 08:30:22 2008 Subject: Plasmid elution from filter problem References: Message-ID: <24NAk.10744$PS3.10546@newsfe06.iad> In , DK wrote: > In article , Nick Theodorakis wrote: >>On Sep 17, 3:40=A0pm, Daniel Prieto wrote: >>> Great info! Didn't know the type of paper was that critical. I will try >>> the high salt elution. Thank you! >>> Daniel >> >>DE81 is not just plain paper -- it's ion-exhange paper. > > Is it even still made? I vaguely remember years ago > a thread dealing with DEAE membrane/paper being > discontinued? It is. -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From sayasatheesh from gmail.com Fri Sep 19 06:44:40 2008 From: sayasatheesh from gmail.com (satheesh kumar) Date: Fri Sep 19 08:32:33 2008 Subject: header intact Message-ID: <135550be0809190444x2f1bc02are9ff5c34ee685f8d@mail.gmail.com> Sir, Im satheesh from India. Im doing work on finding a genetic diversity on different coffea species using RAPD marker analysis. For the RAPD analysis require a Taq Polymerase enzyme. We better to optimization of coffea genetic variability finding purpose, we require a Taq polymerase isolation from recombinent E.coli. So, u suggest me a Taq polymerase isolation protocol with buffer composition volumes. Im waiting for ur reply to me... Thanking u... Satheesh Kumar Junior Research Fellow, Central Coffee Research Institute, India. From shenq from purdue.edu Fri Sep 19 09:42:32 2008 From: shenq from purdue.edu (Qingwu Shen) Date: Fri Sep 19 14:50:08 2008 Subject: Problems with protein expression Message-ID: <1221835352.48d3ba58f2eda@webmail.purdue.edu> Take and others, Here is more detailed information. The vector I'm using is pET-3d, ampicillin resistant gene,T7 promoter. After I harvested the cells, I sonicated them in 25 mM Tris/pH 7.5, 5 mM EDTA, 1 mM DTT, and 8 M urea. Urea was used to dissolve the protein I was trying to express (TnI). After centrifuge (32,000 X g) to remove cell debris, I mixed the supernatant with SDS-PAGE loading buffer (containing 8 M urea) and the load samples on to gel. I think there should be no problem about protein extraction because I used the same protocol to extract both human cardiac TnI and wild type rabbit skeletal TnI. It works for both. Only after I mutated the rabbit skeletal TnI using PCR, I got no protein I wanted. Somebody suggests to re-clone the gene to another vector, do you guy think this will really works? What really confuses is that the wild type can be expressed, but the mutant can not. Anybody can explain this? Your help are really highly appreciated. Best regards, Shen - From wenuganen from yahoo.com Fri Sep 19 10:46:15 2008 From: wenuganen from yahoo.com (wenu ganen) Date: Fri Sep 19 14:50:19 2008 Subject: Trapeze telomerase kit problem Message-ID: <521265.71858.qm@web56204.mail.re3.yahoo.com> Dear Rob Jordan, Firstly , Let me introduce my self. My name : Wenuganen.I am a PhD student in Physiology at Maharishi University of Management , Fairfield Iowa, USA. I am preparing my research on Telomerase activity. I plan to use Trapeze kit.After reading your comment on Trapeze telomerase kit problem I have to consider it again. I think you are so experienced to do this kind of assay. I hope you can advise me and share your experience. I am also looking for some possibilities to get some training to master the telomerase detection system. would you please let me? and please let me know what I have prepare. Thank you very much for your kind attention. All the Best Wenu From tfitzwater from somalogic.com Fri Sep 19 11:21:31 2008 From: tfitzwater from somalogic.com (Tim Fitzwater) Date: Fri Sep 19 14:51:59 2008 Subject: Herring Sperm DNA Message-ID: <771BF0D090CE73449F7495D31E0BA0D12181EB@ELM.sladmin.com> Does anyone know of raw material vendor for sodium salt of herring sperm dna. Sigma is on backorder and I am looking to see if there are any other vendors of this seasonal item. Solomon Dhandu Trevigen sells salmon and herring sperm DNA. http://www.trevigen.com/molecularbiology/hybridDNA.php Tim From novalidaddress from nurfuerspam.de Sat Sep 20 16:40:30 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Sat Sep 20 18:06:48 2008 Subject: [OT?] About Planning a LIMS Message-ID: <6ae2253f-05b9-4264-ad0f-d78666ace5f8@d45g2000hsc.googlegroups.com> Dear experts, I know this post might be considered a bit off topic for this NG, but as I feel quite a bit like home here, I dare to start my research here. I am in charge of setting up a laboratory data management system (LIMS) for a new lab. From hard- and software, I may quite freely choose while it should not be too expensive in the beginning. I am aware that there is a lot of possible solutions on the market, but yet I have no real good point where I could start from. When feasible, I'd prefer open source. That's why I'd like to ask you for some guidance, please! The desired system needs to integrate people (concepts, lab notebooks, reports, references, secure (encrypted) email) with SOPs, equipment (data, status, alarms) and should smoothly integrate into a server based network including access from remote locations. It should be scalable (we start with a small lab and 5 people) and be expandable to manage stock and manufacturing later, meaning, it must be able to grow with a company. Of course the data management must fulfill the requirements for patent applications etc. At this step, I am looking for hints on basic concepts (reviews, web pages, books), as well as material about possible topologies and by what technical means (field bus? LAN? ) to connect lab machines like photometers and refrigerators to the computer system. I'd highly appreciate if someone would like to share a personal experience of such an endeavor! Thousand thanks and best regards! Wolfgang From eenigoy from gmail.com Mon Sep 22 00:05:48 2008 From: eenigoy from gmail.com (yoginee budhkar) Date: Mon Sep 22 12:38:14 2008 Subject: Setting up a new mole biol laboratory Message-ID: <77ddf6c70809212205m6b8e8c71t545f237191342316@mail.gmail.com> Dear Friends, We are planning to set up a molecular biology laboratory from scratch. I am required to make a list of equipments required for regular use in the laboratory. We have already shortlisted major equipment like chemidoc system, RT PCR, 2D gel electrophoresis system, laminar air flow, etc with smaller equipments like submarine gel electrophoresis units, PAGE units, western and southern blot apparatus, speed vac, hand held uv torch, uv transilluminator, tabletop centrifuge, autoclave, and other everyday use apparetus like waterbath, pH meter, magnetic stirrers, vortex, incubator, oven, digital balance and the freezers of -20 and -70 degrees Celsius etc I need some inputs on whether there is anything crucial from the point of view of a mole biol lab that I may have missed out... Any and all suggestions are welcome! Regards, --Yg From darshini.prasanna from gmail.com Mon Sep 22 04:50:37 2008 From: darshini.prasanna from gmail.com (darshini.prasanna@gmail.com) Date: Mon Sep 22 12:38:44 2008 Subject: About PEG Message-ID: <09d6802f-2298-4947-bde8-1dc1db7c802a@p31g2000prf.googlegroups.com> Hi does anyone know how PEG can be used for cell lysis.? whats the mechanism of cell lysis by PEG? From darshini.prasanna from gmail.com Mon Sep 22 04:56:55 2008 From: darshini.prasanna from gmail.com (darshini.prasanna@gmail.com) Date: Mon Sep 22 12:38:50 2008 Subject: neural networks Message-ID: <811b5d78-dfcc-4bdd-9a26-b7cdf69a8727@n33g2000pri.googlegroups.com> hi can anyone help me how to use neural network in matlab? like how to give inputs,weights,targets.? From novalidaddress from nurfuerspam.de Mon Sep 22 16:39:14 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Mon Sep 22 16:56:12 2008 Subject: Setting up a new mole biol laboratory References: Message-ID: <9292eb86-dbbc-4842-a497-76ac7a6f5bcd@l42g2000hsc.googlegroups.com> Dear Yg, basically, you already have mentioned all you need. It might be a good thing to think of the regulations for work with GMOs (which standards to follow to please administration and government) and to apply for necessary permits before you start, however. Another point is to set up a system for tracking plasmids, primers and GMOs (a small homebrew database with eg. Filemaker or something similar should do it). The most important ingredient might be a senior postdoc or technician who is really familiar with molecular biology, knows all the nasty tricks and how to avoid overpriced nice looking boxes containing 'super duper' kits for things that may be achieved much cheaper and faster with homebrew methods and reagents (you might browse our archives here and ask the wise guys of this NG any time). One thing you maybe have omitted in your list is a well assorted set of basic enyzmes and chemicals. Some vendors have special offers for starting labs, so it might be worth to ask your favorite supplier for a decent discount on that what you need. Also access to a source of crushed ice and a quantitative realtime cycler (depending on your scientific tasks) might be a plus. Also do not forget a household microwave oven to boil agarose. A laminar flow actually is not really necessary at all, at least as long as you do not need to protect yourself from your bugs or work in an environment full of molds and dust. If you should want to perform sensitive PCRs or even diagnostics, strongly consider separate rooms for assembling PCRs and running/ analyzing them. Regarding geldocs systems, a cheap digital camera, equipped with an orange filter should and a close-up lens do the job as well. For cloning purposes, instead of a UV transilluminator, you better use a blue LED light source. UV nicely kills DNA almost instantly. You may get a small one for quite cheap together with a box of pre-cast agarose gels in an orange plastic box which makes up a perfect filter for EtBr an CyBrgreen (when cut in pieces. Thinking of Cambrex, now part of Lonza). As an alternative to a water bath, thermocyclers so nicely may be "abused" as programmable incubators... What you won't need is a 2D gel system (at least for ordinary cloning etc.) Best of luck and happy cloning! Wolfgang From novalidaddress from nurfuerspam.de Mon Sep 22 16:58:49 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Tue Sep 23 11:31:25 2008 Subject: neural networks References: <811b5d78-dfcc-4bdd-9a26-b7cdf69a8727@n33g2000pri.googlegroups.com> Message-ID: <7dca9ac8-1eec-4838-a45c-5550e70f38c2@79g2000hsk.googlegroups.com> Dear Darshini, better post this question in the appropriate forum from Mathworks or Octave (a free Matlab clone). https://www-old.cae.wisc.edu/mailman/listinfo/help-octave Enjoy! Wo From SBrown from ccia.unsw.edu.au Mon Sep 22 17:58:19 2008 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Tue Sep 23 11:31:31 2008 Subject: RACE Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A04C54F8B@mail01.ccia.local> Dpes anyone know why the annealing temperatures for 3'RACE are always much higher than normal? S 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 pjie2 from cam.ac.uk Tue Sep 23 11:39:04 2008 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Tue Sep 23 13:42:23 2008 Subject: RACE In-Reply-To: References: Message-ID: When doing a RACE reaction, you need to use high specificity primers with a higher annealing temperature than usual, since you're only using one gene-specific primer in the PCR (along with the adaptor primer). The RACE kit, adaptor primers etc. are thus optimised for this. If you're doing two rounds of amplification, then the second round of PCR can use primers with a lower Tm, as the first round should have already increased the representation of your gene of interest. Peter Ellis On Tue, 23 Sep 2008, Scott Brown wrote: > > Dpes anyone know why the annealing temperatures for 3'RACE are always > much higher than normal? > > S > > > 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 hutsugi from fhcrc.org Tue Sep 23 12:50:34 2008 From: hutsugi from fhcrc.org (Utsugi, Heidi K) Date: Tue Sep 23 13:42:30 2008 Subject: molecular biology laboratory from scratch RE: Methods Digest, Vol 40, Issue 17 References: <200809231705.m8NH5mV28008@net.bio.net> Message-ID: One the most important Mol. Bio. Lab. tool that I tell my graduating Lab Managers (PI newbies) is SPACE FOR A SECURED -80C. Buy a freezer, plug it into a 'dedicated outlet with alarm phone notification' and never, never give any shelf space away. Fill the freezer with empty boxes to give the illusion that it is all in use. You'll use it eventually. Heidi ________________________________ From: methods-bounces@oat.bio.indiana.edu on behalf of methods-request@oat.bio.indiana.edu Sent: Tue 9/23/2008 10:05 AM To: methods@magpie.bio.indiana.edu Subject: Methods Digest, Vol 40, Issue 17 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. Setting up a new mole biol laboratory (yoginee budhkar) 2. About PEG (darshini.prasanna@gmail.com) 3. neural networks (darshini.prasanna@gmail.com) 4. Re: Setting up a new mole biol laboratory (WS) 5. Re: neural networks (WS) 6. RACE (Scott Brown) ---------------------------------------------------------------------- Message: 1 Date: Mon, 22 Sep 2008 10:35:48 +0530 From: "yoginee budhkar" Subject: Setting up a new mole biol laboratory To: methods@magpie.bio.indiana.edu Message-ID: <77ddf6c70809212205m6b8e8c71t545f237191342316@mail.gmail.com> Content-Type: text/plain; charset=ISO-8859-1 Dear Friends, We are planning to set up a molecular biology laboratory from scratch. I am required to make a list of equipments required for regular use in the laboratory. We have already shortlisted major equipment like chemidoc system, RT PCR, 2D gel electrophoresis system, laminar air flow, etc with smaller equipments like submarine gel electrophoresis units, PAGE units, western and southern blot apparatus, speed vac, hand held uv torch, uv transilluminator, tabletop centrifuge, autoclave, and other everyday use apparetus like waterbath, pH meter, magnetic stirrers, vortex, incubator, oven, digital balance and the freezers of -20 and -70 degrees Celsius etc I need some inputs on whether there is anything crucial from the point of view of a mole biol lab that I may have missed out... Any and all suggestions are welcome! Regards, --Yg ------------------------------ Message: 2 Date: Mon, 22 Sep 2008 02:50:37 -0700 (PDT) From: darshini.prasanna@gmail.com Subject: About PEG To: methods@net.bio.net Message-ID: <09d6802f-2298-4947-bde8-1dc1db7c802a@p31g2000prf.googlegroups.com> Content-Type: text/plain; charset=ISO-8859-1 Hi does anyone know how PEG can be used for cell lysis.? whats the mechanism of cell lysis by PEG? ------------------------------ Message: 3 Date: Mon, 22 Sep 2008 02:56:55 -0700 (PDT) From: darshini.prasanna@gmail.com Subject: neural networks To: methods@net.bio.net Message-ID: <811b5d78-dfcc-4bdd-9a26-b7cdf69a8727@n33g2000pri.googlegroups.com> Content-Type: text/plain; charset=ISO-8859-1 hi can anyone help me how to use neural network in matlab? like how to give inputs,weights,targets.? ------------------------------ Message: 4 Date: Mon, 22 Sep 2008 14:39:14 -0700 (PDT) From: WS Subject: Re: Setting up a new mole biol laboratory To: methods@net.bio.net Message-ID: <9292eb86-dbbc-4842-a497-76ac7a6f5bcd@l42g2000hsc.googlegroups.com> Content-Type: text/plain; charset=ISO-8859-1 Dear Yg, basically, you already have mentioned all you need. It might be a good thing to think of the regulations for work with GMOs (which standards to follow to please administration and government) and to apply for necessary permits before you start, however. Another point is to set up a system for tracking plasmids, primers and GMOs (a small homebrew database with eg. Filemaker or something similar should do it). The most important ingredient might be a senior postdoc or technician who is really familiar with molecular biology, knows all the nasty tricks and how to avoid overpriced nice looking boxes containing 'super duper' kits for things that may be achieved much cheaper and faster with homebrew methods and reagents (you might browse our archives here and ask the wise guys of this NG any time). One thing you maybe have omitted in your list is a well assorted set of basic enyzmes and chemicals. Some vendors have special offers for starting labs, so it might be worth to ask your favorite supplier for a decent discount on that what you need. Also access to a source of crushed ice and a quantitative realtime cycler (depending on your scientific tasks) might be a plus. Also do not forget a household microwave oven to boil agarose. A laminar flow actually is not really necessary at all, at least as long as you do not need to protect yourself from your bugs or work in an environment full of molds and dust. If you should want to perform sensitive PCRs or even diagnostics, strongly consider separate rooms for assembling PCRs and running/ analyzing them. Regarding geldocs systems, a cheap digital camera, equipped with an orange filter should and a close-up lens do the job as well. For cloning purposes, instead of a UV transilluminator, you better use a blue LED light source. UV nicely kills DNA almost instantly. You may get a small one for quite cheap together with a box of pre-cast agarose gels in an orange plastic box which makes up a perfect filter for EtBr an CyBrgreen (when cut in pieces. Thinking of Cambrex, now part of Lonza). As an alternative to a water bath, thermocyclers so nicely may be "abused" as programmable incubators... What you won't need is a 2D gel system (at least for ordinary cloning etc.) Best of luck and happy cloning! Wolfgang ------------------------------ Message: 5 Date: Mon, 22 Sep 2008 14:58:49 -0700 (PDT) From: WS Subject: Re: neural networks To: methods@net.bio.net Message-ID: <7dca9ac8-1eec-4838-a45c-5550e70f38c2@79g2000hsk.googlegroups.com> Content-Type: text/plain; charset=ISO-8859-1 Dear Darshini, better post this question in the appropriate forum from Mathworks or Octave (a free Matlab clone). https://www-old.cae.wisc.edu/mailman/listinfo/help-octave Enjoy! Wo ------------------------------ Message: 6 Date: Tue, 23 Sep 2008 08:58:19 +1000 From: "Scott Brown" Subject: RACE To: Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A04C54F8B@mail01.ccia.local> Content-Type: text/plain; charset="us-ascii" Dpes anyone know why the annealing temperatures for 3'RACE are always much higher than normal? S 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. ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 40, Issue 17 *************************************** From rory.obrien from stonebow.otago.ac.nz Tue Sep 23 18:26:58 2008 From: rory.obrien from stonebow.otago.ac.nz (Rory O'Brien) Date: Wed Sep 24 14:02:44 2008 Subject: Setting up a new mole biol laboratory In-Reply-To: <77ddf6c70809212205m6b8e8c71t545f237191342316@mail.gmail.com> Message-ID: A set of Current Protocols and/or the new(er) edition of Maniatis. and an ultrapure water supply, Milli-Q or similar. - R. > Dear Friends, > > We are planning to set up a molecular biology laboratory from > scratch. I am required to make a list of equipments required > for regular use in the laboratory. We have already > shortlisted major equipment like > > chemidoc system, RT PCR, 2D gel electrophoresis system, > laminar air flow, etc > > with smaller equipments like submarine gel electrophoresis > units, PAGE units, western and southern blot apparatus, speed > vac, hand held uv torch, uv transilluminator, tabletop > centrifuge, autoclave, > > and other everyday use apparetus like waterbath, pH meter, > magnetic stirrers, vortex, incubator, oven, digital balance > and the freezers of -20 and -70 degrees Celsius etc > > I need some inputs on whether there is anything crucial from > the point of view of a mole biol lab that I may have missed > out... Any and all suggestions are welcome! > > Regards, > --Yg > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods From zalmemar from uccs.edu Tue Sep 23 18:50:56 2008 From: zalmemar from uccs.edu (Zamawang Faisel Almemar-Ray) Date: Wed Sep 24 14:02:51 2008 Subject: delipidized BCS (FBS) Message-ID: Could someone please tell me how to make delipidized BCS (FBS) using charcoal stripping. I desparately need the procedure for this, as my research is on hold because my order for delipidized BCS (FBS) is on back-order. Thanks, Z From lauredastarac from gmail.com Wed Sep 24 08:55:04 2008 From: lauredastarac from gmail.com (laure d'astarac) Date: Wed Sep 24 14:02:56 2008 Subject: Transfection myoblast Message-ID: Hi, I have to start transfection of AON and siRNA in my lab, where nobody has experience... So several questions about myoblast transfection with small RNAs (or AONs...) 1/ what is the best transfection method to use: Electroporation Vs Lipfectamin Vs other? 2/ If someone has general rules about minimum and maximum amount of AON -Si RNA to use in transfection, in order to be efficient but not toxic... I suppose i have to do several quantities, but values are arround ng, microg??? 3/ When harrest cells after transfection: 24h? 48? test both? I am sorry about these basic questions.... and thank you for your help.... -- Claire Navarro Stem cell laboratory University of Milan - Ospedale Maggiore Policlino di Milano - Padiglione Ponti, II piano Via F. Sforza, 35 20122 Milan Italy Tel: 00 39 02 55 03 38 52 00 39 02 55 03 38 74 From tk from shaggy.csail.mit.edu Thu Sep 25 20:11:36 2008 From: tk from shaggy.csail.mit.edu (Tom Knight) Date: Fri Sep 26 13:37:37 2008 Subject: delipidized BCS (FBS) References: Message-ID: "Zamawang Faisel Almemar-Ray" writes: > Could someone please tell me how to make delipidized BCS (FBS) using > charcoal stripping. I desparately need the procedure for this, as my > research is on hold because my order for delipidized BCS (FBS) is on > back-order. Chen, R. F. (1967). Removal of fatty acids from serum albumin by charcoal treatment. J. Biol. Chem. 242, 173-181. You won't like the complexity. From tk from shaggy.csail.mit.edu Thu Sep 25 20:12:33 2008 From: tk from shaggy.csail.mit.edu (Tom Knight) Date: Fri Sep 26 13:39:02 2008 Subject: delipidized BCS (FBS) References: Message-ID: "Zamawang Faisel Almemar-Ray" writes: > Could someone please tell me how to make delipidized BCS (FBS) using > charcoal stripping. I desparately need the procedure for this, as my > research is on hold because my order for delipidized BCS (FBS) is on > back-order. Chen, R. F. (1967). Removal of fatty acids from serum albumin by charcoal treatment. J. Biol. Chem. 242, 173-181. You won't like the complexity of this process. From virashkgupta from gmail.com Fri Sep 26 05:42:45 2008 From: virashkgupta from gmail.com (Virash Gupta) Date: Fri Sep 26 13:39:31 2008 Subject: Methods Digest, Vol 40, Issue 19, Setting up a new mole biol laboratory Message-ID: Add a good quality laminar flow bench, simple PCR mchines, dedicated microcentrifuge ets as well On 9/25/08, 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: Setting up a new mole biol laboratory (Rory O'Brien) > 2. Transfection myoblast (laure d'astarac) > 3. delipidized BCS (FBS) (Zamawang Faisel Almemar-Ray) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 24 Sep 2008 11:26:58 +1200 > From: "Rory O'Brien" > Subject: RE: Setting up a new mole biol laboratory > To: > Message-ID: > Content-Type: text/plain; charset="US-ASCII" > > A set of Current Protocols and/or the new(er) edition of Maniatis. > > and an ultrapure water supply, Milli-Q or similar. > > - R. > > > > > Dear Friends, > > > > We are planning to set up a molecular biology laboratory from > > scratch. I am required to make a list of equipments required > > for regular use in the laboratory. We have already > > shortlisted major equipment like > > > > chemidoc system, RT PCR, 2D gel electrophoresis system, > > laminar air flow, etc > > > > with smaller equipments like submarine gel electrophoresis > > units, PAGE units, western and southern blot apparatus, speed > > vac, hand held uv torch, uv transilluminator, tabletop > > centrifuge, autoclave, > > > > and other everyday use apparetus like waterbath, pH meter, > > magnetic stirrers, vortex, incubator, oven, digital balance > > and the freezers of -20 and -70 degrees Celsius etc > > > > I need some inputs on whether there is anything crucial from > > the point of view of a mole biol lab that I may have missed > > out... Any and all suggestions are welcome! > > > > Regards, > > --Yg > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > ------------------------------ > > Message: 2 > Date: Wed, 24 Sep 2008 15:55:04 +0200 > From: "laure d'astarac" > Subject: Transfection myoblast > To: methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Hi, > > I have to start transfection of AON and siRNA in my lab, where nobody has > experience... > > So several questions about myoblast transfection with small RNAs (or > AONs...) > > 1/ what is the best transfection method to use: Electroporation Vs > Lipfectamin Vs other? > > 2/ If someone has general rules about minimum and maximum amount of AON -Si > RNA to use in transfection, in order to be efficient but not toxic... I > suppose i have to do several quantities, but values are arround ng, > microg??? > > 3/ When harrest cells after transfection: 24h? 48? test both? > > I am sorry about these basic questions.... and thank you for your help.... > > -- > Claire Navarro > Stem cell laboratory > University of Milan - Ospedale Maggiore Policlino di Milano - > Padiglione Ponti, II piano > Via F. Sforza, 35 > 20122 Milan > Italy > Tel: 00 39 02 55 03 38 52 > 00 39 02 55 03 38 74 > > > ------------------------------ > > Message: 3 > Date: Tue, 23 Sep 2008 17:50:56 -0600 > From: "Zamawang Faisel Almemar-Ray" > Subject: delipidized BCS (FBS) > To: methods@magpie.bio.indiana.edu > Message-ID: > Content-Type: text/plain;charset=iso-8859-1;format="flowed" > > Could someone please tell me how to make delipidized BCS (FBS) using > charcoal stripping. I desparately need the procedure for this, as my > research is on hold because my order for delipidized BCS (FBS) is on > back-order. > > Thanks, > Z > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 40, Issue 19 > *************************************** > -- 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 sayasatheesh from gmail.com Fri Sep 26 06:11:26 2008 From: sayasatheesh from gmail.com (satheesh kumar) Date: Fri Sep 26 13:39:38 2008 Subject: for estimation of acid phosphatase Message-ID: <135550be0809260411k3d737c29n4e847258f5980a08@mail.gmail.com> hi, Im satheesh kumar, i plan to estimate the acid phosphatase activity in coffea. so anybody wil send me some our good suggestion to me... From SBrown from ccia.unsw.edu.au Fri Sep 26 13:04:05 2008 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Fri Sep 26 13:39:54 2008 Subject: siRNA knockdown and MTT Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A02972A83@mail01.ccia.local> Hi, I am attempting to knockdown a gene then put the cells into an MTT assay for 48 hrs. I need to the knockdown to last the length of the MTT assay. I'm currently trying a 6hr siRNA transfection plated into 96 well plate, then treating with drug and measuring MTT response 48 hrs from that time point. Can anyone suggest any "tricks" either with extending or enhancing knockdown effect or any ammended MTT protocols capable of producing a measurable response. Many Thanks S 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 TieQiao.Wu from bakeridi.edu.au Thu Sep 25 20:48:25 2008 From: TieQiao.Wu from bakeridi.edu.au (TieQiao Wu) Date: Fri Sep 26 13:41:44 2008 Subject: Methods Digest In-Reply-To: <200809251705.m8PH5EV21217@net.bio.net> References: <200809251705.m8PH5EV21217@net.bio.net> Message-ID: <217613C88C00D4448B099AD7A374F25307838DCB@exch01.bhri.internal> Dear All: I am writing to you for getting your help. We recently tried to amplify a 1kb human genomic fragment with 85% GC content and tried different methods, however we failed to get products. Does anyone have any suggestion and advice? Thanks in advance. Tieqiao Charity sponsor of National Ride to Work Day. 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 patrickzhang76 from gmail.com Fri Sep 26 15:07:57 2008 From: patrickzhang76 from gmail.com (patrickzhang76@gmail.com) Date: Fri Sep 26 17:49:40 2008 Subject: retroviral vs lentiviral expression Message-ID: Hi, there. For the purpose of overexpressing a gene of interest in the dividing mammalian cells, is there any difference between using a retroviral systems and the newer lentiviral systems? Beside the obvious differences for non-dividing cells, what are the pros and cons between the two systems? Thanks. Patrick From shurjo.sen from gmail.com Fri Sep 26 15:17:23 2008 From: shurjo.sen from gmail.com (Shurjo Kumar Sen) Date: Fri Sep 26 17:49:53 2008 Subject: Fwd: Methods Digest, Vol 40, Issue 19, Setting up a new mole biol laboratory In-Reply-To: References: Message-ID: <310d0a160809261317y42a685d6g7825be2d66da2091@mail.gmail.com> A Nanodrop benchtop spectrophotometer is a good idea. Also, check out the newest generation of transilluminators which use blue light and are much safer than UV for both users and DNA. They work really well (I just used one yesterday). http://www.clarechemical.com/transilluminator.htm ---------- Forwarded message ---------- From: Virash Gupta Date: 2008/9/26 Subject: Re: Methods Digest, Vol 40, Issue 19, Setting up a new mole biol laboratory To: methods@oat.bio.indiana.edu Add a good quality laminar flow bench, simple PCR mchines, dedicated microcentrifuge ets as well On 9/25/08, 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: Setting up a new mole biol laboratory (Rory O'Brien) > 2. Transfection myoblast (laure d'astarac) > 3. delipidized BCS (FBS) (Zamawang Faisel Almemar-Ray) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 24 Sep 2008 11:26:58 +1200 > From: "Rory O'Brien" > Subject: RE: Setting up a new mole biol laboratory > To: > Message-ID: > Content-Type: text/plain; charset="US-ASCII" > > A set of Current Protocols and/or the new(er) edition of Maniatis. > > and an ultrapure water supply, Milli-Q or similar. > > - R. > > > > > Dear Friends, > > > > We are planning to set up a molecular biology laboratory from > > scratch. I am required to make a list of equipments required > > for regular use in the laboratory. We have already > > shortlisted major equipment like > > > > chemidoc system, RT PCR, 2D gel electrophoresis system, > > laminar air flow, etc > > > > with smaller equipments like submarine gel electrophoresis > > units, PAGE units, western and southern blot apparatus, speed > > vac, hand held uv torch, uv transilluminator, tabletop > > centrifuge, autoclave, > > > > and other everyday use apparetus like waterbath, pH meter, > > magnetic stirrers, vortex, incubator, oven, digital balance > > and the freezers of -20 and -70 degrees Celsius etc > > > > I need some inputs on whether there is anything crucial from > > the point of view of a mole biol lab that I may have missed > > out... Any and all suggestions are welcome! > > > > Regards, > > --Yg > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > ------------------------------ > > Message: 2 > Date: Wed, 24 Sep 2008 15:55:04 +0200 > From: "laure d'astarac" > Subject: Transfection myoblast > To: methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Hi, > > I have to start transfection of AON and siRNA in my lab, where nobody has > experience... > > So several questions about myoblast transfection with small RNAs (or > AONs...) > > 1/ what is the best transfection method to use: Electroporation Vs > Lipfectamin Vs other? > > 2/ If someone has general rules about minimum and maximum amount of AON -Si > RNA to use in transfection, in order to be efficient but not toxic... I > suppose i have to do several quantities, but values are arround ng, > microg??? > > 3/ When harrest cells after transfection: 24h? 48? test both? > > I am sorry about these basic questions.... and thank you for your help.... > > -- > Claire Navarro > Stem cell laboratory > University of Milan - Ospedale Maggiore Policlino di Milano - > Padiglione Ponti, II piano > Via F. Sforza, 35 > 20122 Milan > Italy > Tel: 00 39 02 55 03 38 52 > 00 39 02 55 03 38 74 > > > ------------------------------ > > Message: 3 > Date: Tue, 23 Sep 2008 17:50:56 -0600 > From: "Zamawang Faisel Almemar-Ray" > Subject: delipidized BCS (FBS) > To: methods@magpie.bio.indiana.edu > Message-ID: > Content-Type: text/plain;charset=iso-8859-1;format="flowed" > > Could someone please tell me how to make delipidized BCS (FBS) using > charcoal stripping. I desparately need the procedure for this, as my > research is on hold because my order for delipidized BCS (FBS) is on > back-order. > > Thanks, > Z > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 40, Issue 19 > *************************************** > -- Dr V K Gupta Sr Microbiologist (Molecular Biology) Insect Molecular Biology Lab Department of Entomology Punjab Agricultural University Ludhiana (Pb)-141004- India M: 09815963210 _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods -- ********************************************************** Shurjo Kumar Sen, Ph.D. Postdoctoral Visiting Fellow Genome Technology Branch National Human Genome Research Institute National Institutes of Health Building 50, Room 5529 50 South Drive, MSC 8004 Bethesda, MD 20892-8004 Phone: 225-281-6808 e-mail: sensh@mail.nih.gov From novalidaddress from nurfuerspam.de Fri Sep 26 16:07:25 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Fri Sep 26 17:51:02 2008 Subject: for estimation of acid phosphatase References: Message-ID: <9f7eb190-38b8-48c6-b002-31d5cc13b1bf@k13g2000hse.googlegroups.com> Hi, cheapest way should be pNPP (p-nitrophenyl phosphate) as substrate. If you prefer higher sensitivity and/or prefer fluorescent detection, you might choose MUPF (methyl umbelliferyl phosphate) or ELF phosphate, just to mention some possibilities. If you want to use chemiluminescence, check CSPD/CPD-star substrates. pH is adjusted with appropriate buffer in all cases. Best reagrds, Wo On Sep 26, 1:11?pm, "satheesh kumar" wrote: > hi, > > ? ?Im satheesh kumar, i plan to estimate the acid phosphatase activity in > coffea. so anybody wil send me some our good suggestion to me... From aawara from pontiff-playground.org Fri Sep 26 22:17:57 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Sat Sep 27 13:06:51 2008 Subject: retroviral vs lentiviral expression References: Message-ID: In , patrickzhang76@gmail.com wrote: > Hi, there. For the purpose of overexpressing a gene of interest in the > dividing mammalian cells, is there any difference between using a > retroviral systems and the newer lentiviral systems? Beside the > obvious differences for non-dividing cells, what are the pros and cons > between the two systems? Retroviral vectors work extremely well for dividing cells. There are fewer viral proteins that need to be expressed, and typically support larger inserts. Although MLV vectors are restricted to a total size of about 8 kb, I've used Steve Hughes' avian retrovirus vectors with a total size of about 11 kb, of which about 8.5 kb was the insert ...... AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From tk from shaggy.csail.mit.edu Fri Sep 26 22:18:59 2008 From: tk from shaggy.csail.mit.edu (Tom Knight) Date: Sat Sep 27 13:07:00 2008 Subject: Methods Digest References: <200809251705.m8PH5EV21217@net.bio.net> Message-ID: "TieQiao Wu" writes: > I am writing to you for getting your help. We recently tried to amplify > a 1kb human genomic fragment with 85% GC content and tried different > methods, however we failed to get products. Does anyone have any > suggestion and advice? Thanks in advance. I'd suggest adding 5-8% 1 molar betaine (Sigma) to your PCR reaction. You could also try the Phusion high GC master mix (Finnzyme, NEB). From manuelg from telus.net Sun Sep 28 18:49:37 2008 From: manuelg from telus.net (Manuel Gadin) Date: Mon Sep 29 08:59:14 2008 Subject: missing bands and ghost bands Message-ID: <57D222FB5DA743D5A100C29238845814@ManuelPC> Hello. The method/procedure , master mix , gels and thermocycler that I am using is the same that my co-lab workers are using. Having said that, my techniques could be the only culprit. I am the only one that cannot get consistent results. I am failry new to PCR.Sometimes, I would be missing bands in my positive control and also I would get ghost bands. Few of my co workers had watched me run the procedure and the only comment I got was maybe the parafilm degraded my DNA bands. The parafilm would sometimes stick to my pipet tips when I am mixing the samples with the loading dye.Other than that, they cannot offer any solution. Help!! I have been trying to get consistent results for 8 monthss but to no avail!!! Desperate PCR lab worker From editor from gene-quantification.info Mon Sep 29 05:06:41 2008 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Mon Sep 29 08:59:46 2008 Subject: qPCR Newsletter September 2008 Message-ID: <0233a41a-3b0b-414a-b65c-7d6791308714@k37g2000hsf.googlegroups.com> qPCR Newsletter September 2008 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: - microRNA - new links and animations - microRNA - updated microRNA application papers - worldwide qPCR application workshops - qPCR Symposium USA in November 2008 - register now ! - 1st announcement qPCR 2009 Event =3D> http://www.qPCR2009.net ---------------------------------------------------------------------------= ----- New http://www.Gene-Quantification.ASIA pages online translation to CHINESE =3D> http://chinese.gene-quantification.asia/ JAPANESE =3D> http://japanese.gene-quantification.asia/ KOREAN =3D> http://korean.gene-quantification.asia/ RUSSIAN =3D> http://russian.gene-quantification.asia/ ---------------------------------------------------------------------------= ----- Links on http://microrna.gene-quantification.info/ microRNA Definition non-coding RNAs / DataBase micro RNA talks and microRNA posters Nature Genetics 38, S1 (2006) - The microRevolution miRNA Research Education Research Center Human MicroRNA targets miRU - Plant microRNA Potential Target Finder Cancer genomics: Small RNAs with big impacts miRU: an automated plant miRNA target prediction server miRBase - the home of microRNA data miRBase: microRNA sequences, targets and gene nomenclature The microRNA Registry A uniform system for microRNA annotation TRC - the RNAi Consortium shRNA Library Algorithms & Thermodynamics for Nucleic Acid Folding, Hybridization & Melting Profile Prediction TUTORIAL: Check out an illustrated tutorial on miRNA (by Ambion) ---------------------------------------------------------------------------= ----- A lot of new application papers using microRNA quantitative real-time RT-PCR http://microRNA.gene-quantification.info Strategies to determine the biological function of microRNAs. Genomics of microRNA. Tracing microRNA patterns in mice. Target mimics modulate miRNAs. Target mimicry provides a new mechanism for regulation of microRNA activity. Lessons from Nature: microRNA-based shRNA libraries. Identification of Differentially Expressed MicroRNAs by Microarray: A Possible Role for MicroRNA Genes in Pituitary Adenomas. RNA polymerase III transcribes human microRNAs. Real-time expression profiling of microRNA precursors in human cancer cell lines. Multiplexing RT-PCR for the detection of multiple miRNA species in small samples. An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. MicroRNA expression profiling using LNA-modified probes in a liquid- phase bead-based array. miRCURY=99 LNA research tools for microRNA. Editorial - The microRevolution. Glimpses of a Tiny RNA World. Functions of microRNAs and related small RNAs in plants. Evidence that microRNAs are associated with translating messenger RNAs in human cells. How microRNAs control cell division, differentiation and death. microRNA target predictions in animals. Human let-7a miRNA blocks protein production on actively translating polyribosomes. Computational identification of microRNA targets. Intronic microRNA precursors that bypass Drosha processing. MicroInspector: a web tool for detection of miRNA binding sites in an RNA sequence. Target labelling for the detection and profiling of microRNAs expressed in CNS tissue using microarrays. A brain-specific microRNA regulates dendritic spine development. ---------------------------------------------------------------------------= ----- Update of the Webinar page A lot of interesting TALKs, WEBINARs, SLIDE SHOWs, and PODCASTs from various speakers, biotec companies, qPCR Events, and international journals (Nature and Science) are FREE for download. Have a look and you will definitely something interesting for your scientific work ! http://webinar.gene-quantification.info/ ---------------------------------------------------------------------------= ----- With the new qPCR INFO PORTAL and all the presented tools we will help you with to find the right information about qPCR and related topics in Molecular Biology in the literature and in the World Wide Web. =3D> Papers / Protocols / Methods / Databases / Alets / Feeds / Books / Forums / E-mail / Directory http://infoportal.gene-quantification.info/ ---------------------------------------------------------------------------= ----- 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 =3D> events@gene- quantification.info ---------------------------------------------------------------------------= ----- qPCR SYMPOSIUM BENELUX The prominent and still growing place taken by real-time quantitative PCR in applied and fundamental research and clinical diagnostics almost appears obvious. However, it is clear that contributions made by various scientists and companies in the field during the last decade rendered this technology useful and affordable for many users. More info =3D> http://www.gene-quantification.de/meetings.html#benelux Importantly, the qPCR domain is still in constant evolution, making it sometimes hard to stay informed about new methodological approaches or original studies using the real-time PCR. Therefore, we have scheduled a one day "Benelux qPCR Symposium" on October 6th 2008, giving the opportunity to the scientific community to get informed and discuss various aspects of real-time PCR (including but not limited to new applications, assay optimization and validation, new technologies, etc.). Scientific talks, posters sessions and industrial booths will be at the menu. Download poster =3D> http://www.gene-quantification.de/qpcr_benelux_poster= .pdf ---------------------------------------------------------------------------= ----- qPCR Symposium USA 10. - 13. November 2008 Clarion Hotel San Francisco Airport, Millbrae, CA , USA More info =3D> http://www.gene-quantification.de/meetings.html#qpcr_usa - High throughput platforms: High throughput applications, real-time RT-PCR arrays, digital PCR - Forthcoming technologies: Immuno PCR, Methylation sensitive PCR, SNP analysis, High resolution melt, microRNA detection, - Multiplex technologies - Single-cell qPCR: Pre-amplification techniques, sub-cellular PCR, Expression heterogeneity, laser microdissection, FACS sorting, Enrichment of rare cells - Multimarker diagnostics: Disease markers, Tissue specific markers, Cancer markers, Stem cells, Differentiation markers, Cancer stem cells - Real-time PCR Expression Profiling: multivariate and multiway expression profiling, temporal expression profiling, spatiotemporal maps - Pre-analytical Steps: Sampling technologies, Extraction methods, Reverse Transcription, Quality Control, Standards, Standard Operating Procedures, Interlaboratory Exercises - Normalization & Standardization: Normalization strategies, Reference genes, Spikes, Standard curves, multiplexing, inter-run calibrators, quantification strategies, mRNA degradation - Data management and data treatment: software applications, data mining, data visualization, biostatistics, multivariate statistics ---------------------------------------------------------------------------= ----- qPCR 2009 Event 1st annoncement of the qPCR 2009 Event in Freising Weihenstephan http://www.qPCR2009.net Download flyer =3D> http://www.bioeps.com/qpcr2009/qPCR-2009-1st-announcem= ent.pdf ---------------------------------------------------------------------------= ----- qPCR WORKSHOP TATAA Biocenter Germany - qPCR Application workshops At the TATAA Biocenter Germany we offer qPCR application workshops, the 3-day Core Module and a 2-day Biostatistics Module. qPCR courses are held in regularly in G=F6teborg, 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 courses are held in cooperation with the Institute of Physiology, located at the Technical University of Munich, in Freising-Weihenstephan, near Munich, very close to the Munich Airport (MUC). For more information and to register for the qPCR application workshops, please see our web page: http://tataa.gene-quantification.info/ Course Occasions summer and autumn 2008: 13-17 Oct Prague RNA Isolation + qPCR Core Module + HRM 13-17 Oct Freising Germany qPCR Core Module + Biostatistics (Kurs wird in DEUTSCH gehalten, German language) 27-31 Oct G=F6teborg qPCR Core Module + HRM + Biostatistics 17-21 Nov Prague qPCR Core Module + Practical Biostatistics 24-28 Nov Freising Germany qPCR Core Module + Biostatistics (English language) 1-5 Dec G=F6teborg qPCR Core Module + Biostatistics 15-19 Dec Prague RNA Isolation + Expression Profiling and Data Analysis Download list of TATAA courses in autumn and fall 2008 Please register here =3D> http://www.tataa.com/Courses/Courses.html ---------------------------------------------------------------------------= ----- 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 =A9 2005 - 2008 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 The qPCR newsletter was end to [alle-adressen] 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=3DSUBSCRIBE From prae from gmx.net Tue Sep 30 04:09:19 2008 From: prae from gmx.net (Christian Praetorius) Date: Tue Sep 30 08:52:10 2008 Subject: missing bands and ghost bands References: Message-ID: <6ke8mdF7b3aoU1@mid.individual.net> "Manuel Gadin" wrote: First: Could you please set the length of your line to something like 70 characters? That makes it much easier to read. >The method/procedure , master mix , gels and thermocycler that I am using is the same that my >co-lab workers are using. Having said that, my techniques could be the only culprit. I am the only >one that cannot get consistent results. I am failry new to PCR.Sometimes, I would be missing bands >in my positive control and also I would get ghost bands. Few of my co workers had watched me run >the procedure and the only comment I got was maybe the parafilm degraded my DNA bands. The >parafilm would sometimes stick to my pipet tips when I am mixing the samples with the loading dye. >Other than that, they cannot offer any solution. I don't believe that Parafilm makes any trouble here. I am using the same method for years, and when I had problems, it was always something different, although it is not always easy to find. Please describe your protocol in detail, what is the origin of your DNA and so on. And if possible, show a photo of one of your gels. Christian -- X-no-Sig: yes From higginsb78 from gmail.com Tue Sep 30 09:12:10 2008 From: higginsb78 from gmail.com (Brian P Higgins) Date: Tue Sep 30 17:42:53 2008 Subject: missing bands and ghost bands In-Reply-To: <6ke8mdF7b3aoU1@mid.individual.net> References: <6ke8mdF7b3aoU1@mid.individual.net> Message-ID: One quick question: have any of your coworkers actually tried to run the PCR that you seem to be having problems with? If they can get it to work well, then the problem is your technique. No offense, but 8 months with shoddy PCR results is 7 and a half months too long--someone should have helped you with this LONG ago. You say that your technique is the only culprit, but what about template and primers? You didn't define what constituted your mastermix, so I'm assuming that contains the usual: buffer, Mg, Taq, and dNTPs. Is it possible you miscalculated your primer dilutions and/or template concentration? Just a thought. I agree with Christian--more info would help. good luck! brian On Tue, Sep 30, 2008 at 5:09 AM, Christian Praetorius wrote: > "Manuel Gadin" wrote: > > First: Could you please set the length of your line to something like > 70 characters? That makes it much easier to read. > > >The method/procedure , master mix , gels and thermocycler that I am using > is the same that my > >co-lab workers are using. Having said that, my techniques could be the > only culprit. I am the only > >one that cannot get consistent results. I am failry new to PCR.Sometimes, > I would be missing bands > >in my positive control and also I would get ghost bands. Few of my co > workers had watched me run > >the procedure and the only comment I got was maybe the parafilm degraded > my DNA bands. The > >parafilm would sometimes stick to my pipet tips when I am mixing the > samples with the loading dye. > >Other than that, they cannot offer any solution. > > I don't believe that Parafilm makes any trouble here. I am using the > same method for years, and when I had problems, it was always > something different, although it is not always easy to find. > Please describe your protocol in detail, what is the origin of your > DNA and so on. And if possible, show a photo of one of your gels. > > Christian > > -- > X-no-Sig: yes > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From vpireiner from gmail.com Tue Sep 30 10:32:16 2008 From: vpireiner from gmail.com (Becky Pickin) Date: Tue Sep 30 17:43:01 2008 Subject: PCR help please Message-ID: Hello all, I am back again this time asking for help with some problem PCR. I will do my best to explain what I have done. If you have any suggestions or tips/tricks I am quite willing to give them a try. I have tested every piece I can think of and have run out of ideas...none of my PCR reactions have resulted in a band - specific or not. All products should be between 200bp and 1200bp. (negative controls are always blank - no streak, smear, band..nothing - very clean) Let me start with what I'm doing: PCR on HeLa cell genomic DNA both EcoRI digested and not. The initial template used was <1 yr old HeLa genomic DNA isolated in our lab by another member (excellent lab hands) and digested with EcoRI. He has used this template in PCR and for probe design within the past 6 months. Concentration has been re-checked using a BioRad Spec measuring A260/280. In addition, dilutions (down to the concentration used in PCR reactions) of the DNA were run on a gel and resulted in the predicted smear (b/c it was EcoRI digested). Alternative templates were freshly isolated (within the past 2 weeks) HeLa genomic DNA not EcoRI digested and a purified plasmid DNA. All templates have been diluted to ~20ng/ul and 1ul added to the PCR reaction. Templates (except for the undigested genomic) have been used successfully by other lab members. I designed 7 pairs of primers using the Primer 3 program and double checking primer dimers, etc. using the oligo calculator at Northwestern. I have experience designing primers and had another lab member (faculty) double check them before I ordered the primers. Primers are designed to have a Tm of 55-65oC depending on how you calculate (ie salts, etc.) The target Tm was 60oC. Chosen pairs were not anticipated to have any hairpins, primer dimers, etc (I had enough flexibility to select against any that were predicted to be problematic in that regards). These are the "experimental" primers. All primers are reconstituted in TE at 100uM and then diluted to 10uM in autoclaved ddH2O for a working stock. These were ordered and reconstituted within the past 2 weeks. Positive control primers are also at 10uM working stocks and have been shown to give a band with the EcoRI digested HeLa genomic DNA within the week by another lab member (as part of the trouble shooting process) in QPCR. Dissociation curves show a specific product using Sybr Green chemistry. These primers have also been used successfully by other lab members (using more stringent reaction conditions, annealing temp of 60oC) and was why they were selected as a positive control for these experiments. I have been unsuccessful at obtaining a product with these primers using any of the three templates listed above. My reaction chemistry is as follows: * Conc in reaction* *Volume used (ul)/rxn* 10x PCR Buffer (-MgCl2) from Invitrogen 1x 5 50mM MgCl2 1.5mM 1.5 5u/ul Taq Polymerase 1u 0.5 (these are all part of Invitrogen Platinum Taq Polymerase "kit" Cat #10966-026 and are fresh and have been used in a PCR by another lab member and resulted in a positive band) Autoclaved ddH2O - 40 10mM dNTP mix 0.2mM @ 1 Primer 1 0.2uM @ 0.5 Primer 2 0.2uM @ 0.5 Template (diluted to 20ng/ul) 20ng * 1 * 50ul per reaction dNTPs are from Roche diagnostics (#11277049001) shipped as 100mM lithium salt solutions at pH7 (10umol in 100ul). I mixed the 4 dNTPs together as such: 20ul of @dNTP + 120ul ddH2O, made within the past 2 weeks and stored at -20oC. Reactions are set up with a premix adding water followed by buffer first. Some reactions had template added separately, some reactions had primers added separately. Reaction Conditions tried: Try 1: Step 1 94oC 2min 57oC 1min 1 cycle 72oC 2 min Step 2 94oC 1min 57oC 1min 28 cycles 72oC 2min Step 3 94oC 1min 57oC 1min 1 cycle 72oC 10min Step 4 Hold at 4oC Try 2: Same as Try 1 with annealing temperature reduced to 55oC, total cycles increased to 40 Try 3: Step 1 94oC 2min 55oC 1min 1 cycle 72oC 2 min Step 2 94oC 30 sec 55oC 30 sec 38 cycles 72oC 2min Step 3 94oC 30 sec 55oC 30 sec 1 cycle 72oC 10min Step 4 Hold at 4oC 2 different thermocyclers have been tried. Both have a heated lid and thus no oil was applied to the reactions. PCR "products" have loading dye added and then are applied to between 1% and 1.5% agarose gels. The gels are run at 100-120 volts until the dye front migrates ~2/3 of the way down the gel. Gels are then stained with EtBr and photographed on UV light box. Markers used are beautiful. Help please! Thank you, R Pickin, PhD