From ravi1709 from gmail.com Mon Jun 1 04:50:11 2009 From: ravi1709 from gmail.com (ravi1709) Date: Mon Jun 1 08:59:27 2009 Subject: RNA isolation from pigeonpea Message-ID: <08bccbf3-fb8c-481f-bc87-117624de3998@21g2000vbk.googlegroups.com> Hello friends, I want to know the protocol for total RNA isolation from my plant pigeonpea.Whether using kit based isolation will be ok or it will be better to go manual RNA isolation because i dont have any particular protocol for pigeonpea..So, please help me.. Ravi Ranjan From gang.dong from univie.ac.at Mon Jun 1 06:53:26 2009 From: gang.dong from univie.ac.at (Gang Dong) Date: Mon Jun 1 08:59:35 2009 Subject: How to effectively prevent protein aggregation? Message-ID: <000c01c9e2af$930fcc10$b92f6430$@dong@univie.ac.at> Dear all, We are having problems to purify a protein due to aggregation. The protein is MBP-tagged and stays in solution after the tag is removed. Limited proteolysis suggested this protein is mostly folded, with its C-terminal 100 aa unstructured. However, both the full-length and the truncated versions were eluted at the void volume on the S-200 GF column. We suspected that this is due to non-specific interactions among molecules that leads to the aggregation. Since this protein is a component of a large complex, the non-specific interaction might be caused by the exposure of some hydrophobic patches. Before we try out different additives, I am wondering whether any of you have similar experience? If so, how did you solve the problem. Any suggestions are welcome. FYI, the buffer we used contains 20mM Tris (pH8), 300mM NaCl, 5% glycerol. Best, Gang ________________ Gang Dong Max F. Perutz Laboratories (MFPL) Vienna, Austria From novalidaddress from nurfuerspam.de Mon Jun 1 10:19:06 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Mon Jun 1 11:34:50 2009 Subject: How to effectively prevent protein aggregation? References: Message-ID: <4fccd3dd-1fd9-4444-8d73-de20ba2feb8b@c36g2000yqn.googlegroups.com> Dear Gang, try a buffer with a pH as far away as possible from the isoelectric point of your protein and add some non-ionic detergent like TX100 or Tween20. Wo From fengzeng0827 from gmail.com Mon Jun 1 11:54:52 2009 From: fengzeng0827 from gmail.com (feng zeng) Date: Mon Jun 1 12:54:03 2009 Subject: Cytoxicity dose range is too narrow Message-ID: <2a82b3da0906010954m7c20587apff3ccdfa1a389cb3@mail.gmail.com> Is there some people could give me some suggestions about my experiment design I test two compounds' cytoxicity using HT-29 and Zr 75-1 cell line. I set the treatment doses as: 200um,100um,50um,25um,12.5um,6.25um cell seeding at 5x10*3 cells/well, incubated 24h for treatment after adding compound, incubated another 24h then use MTS as detection reagents I got result as: the three high doses (200um, 100um,50um) killed all the cells while the three low doses (25um,25um,12.5um) could not kill any cell. So I calculator the IC50 between 50um and 25um. But why the dose range is so small?? Or this is normal, I just asked a stupid question?? ANY COMMENTS and SUGGESTION are highly appreciated. Thanks to all Feng From shifalich from rediffmail.com Tue Jun 2 21:32:32 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Tue Jun 2 21:38:03 2009 Subject: How to effectively prevent protein aggregation? Message-ID: <1243865256.S.4905.9456.f4mail-235-232.rediffmail.com.old.1243996352.51551@webmail.rediffmail.com> Dear Gong! Aggregate formation can be prevented by addition of Glycerol upto 0.2M. 20% of Glycerol + Tris 20mM should be good enough. Try to avoid Nacl. High salt conc. for some proteins favor aggregation. Tris is good anough. I know 20% glycerol is bit on higher side. But, I have successfully used this buffer for my protein which was pptg. in Tris 50mM, NaCl 150mM, 0.1%Triton X 100. While purification on RP-HPLC, the pressure was crossing limits while injection but, decreasing the flow rate, helped solve the pressure problem. I hope it hepls. Shifali On Mon, 01 Jun 2009 19:37:36 +0530 wrote >Dear all, > > > >We are having problems to purify a protein due to aggregation. The protein >is MBP-tagged and stays in solution after the tag is removed. Limited >proteolysis suggested this protein is mostly folded, with its C-terminal 100 >aa unstructured. However, both the full-length and the truncated versions >were eluted at the void volume on the S-200 GF column. We suspected that >this is due to non-specific interactions among molecules that leads to the >aggregation. Since this protein is a component of a large complex, the >non-specific interaction might be caused by the exposure of some hydrophobic >patches. Before we try out different additives, I am wondering whether any >of you have similar experience? If so, how did you solve the problem. Any >suggestions are welcome. > > > >FYI, the buffer we used contains 20mM Tris (pH8), 300mM NaCl, 5% glycerol. > > > >Best, > >Gang > >________________ > >Gang Dong > >Max F. Perutz Laboratories (MFPL) > >Vienna, Austria > > > > > >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods > Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From rahimpour_a from yahoo.com Wed Jun 3 07:21:27 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Wed Jun 3 07:26:50 2009 Subject: usefull epitope tags in mammalian expression Message-ID: <50724.83797.qm@web52708.mail.re2.yahoo.com> dear all I am going to overexpress some proteins in CHO cell, but I dont have any expreince with usefull epitope tags which I could use for western blot confirmation of expressed proteins, I read about myc, ha, his, flag tags but I am curious to know which one shows better resolution and lower background in mammalian cells. Can anybody help? regards From prae from gmx.net Wed Jun 3 09:27:49 2009 From: prae from gmx.net (Christian Praetorius) Date: Wed Jun 3 09:54:00 2009 Subject: Promoter amplification Message-ID: <78nfj6F1mtj2uU1@mid.individual.net> Hi, I have some trouble with the amplification of a human promoter, which shall be subsequently cloned to a luciferase expression vector. Initially I worked with genomic DNA, which was prepared in our lab and worked quite well for other applications. I finally also got the BAC clone containing the sequence in question. I tried doing PCR with different Mg-concentrations (from 1-4mM), different additives (betaine, DMSO, BSA), different annealing temperaturs as well as running a few cycles with a lower annealing temp. and then using a higher (and combinations of the methods above). The problem with this promoter is a stretch with 90-100% GC-content near to the transcription start site. I get either no product or a bunch of them with much lower sizes than expected, nested PCR gave the same results. Does anyone have any comments or ideas? Christian -- X-no-Sig: yes From pjie2 from cam.ac.uk Wed Jun 3 11:16:10 2009 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Wed Jun 3 12:51:11 2009 Subject: Promoter amplification In-Reply-To: <78nfj6F1mtj2uU1@mid.individual.net> References: <78nfj6F1mtj2uU1@mid.individual.net> Message-ID: Do you have to PCR it, or could you cut a restriction fragment out of a BAC prep and clone that instead? Peter On Wed, 3 Jun 2009, Christian Praetorius wrote: > Hi, > I have some trouble with the amplification of a human promoter, which > shall be subsequently cloned to a luciferase expression vector. > Initially I worked with genomic DNA, which was prepared in our lab and > worked quite well for other applications. I finally also got the BAC > clone containing the sequence in question. > I tried doing PCR with different Mg-concentrations (from 1-4mM), > different additives (betaine, DMSO, BSA), different annealing > temperaturs as well as running a few cycles with a lower annealing > temp. and then using a higher (and combinations of the methods above). > The problem with this promoter is a stretch with 90-100% GC-content > near to the transcription start site. > I get either no product or a bunch of them with much lower sizes than > expected, nested PCR gave the same results. > Does anyone have any comments or ideas? > > Christian > > -- > X-no-Sig: yes > From prae from gmx.net Thu Jun 4 06:55:25 2009 From: prae from gmx.net (Christian Praetorius) Date: Thu Jun 4 11:58:00 2009 Subject: Promoter amplification References: <78nfj6F1mtj2uU1@mid.individual.net> Message-ID: <78pr1fF1krs6cU1@mid.individual.net> Peter Ellis wrote: >Do you have to PCR it, No, I don't have to. Its simply easier, when it works. >or could you cut a restriction fragment out of a >BAC prep and clone that instead? Good sugestion. I am actually digging into it, and it seems possible. But I have to change the MCS of my plasmid but inserting a oligo with the right sites. Christian -- X-no-Sig: yes From aawara from pontiff-playground.org Fri Jun 5 15:48:48 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Fri Jun 5 18:01:23 2009 Subject: Promoter amplification References: <78nfj6F1mtj2uU1@mid.individual.net> <78pr1fF1krs6cU1@mid.individual.net> Message-ID: In <78pr1fF1krs6cU1@mid.individual.net>, Christian Praetorius wrote: > Peter Ellis wrote: > >>Do you have to PCR it, > > No, I don't have to. Its simply easier, when it works. > >>or could you cut a restriction fragment out of a >>BAC prep and clone that instead? > > Good sugestion. I am actually digging into it, and it seems possible. > But I have to change the MCS of my plasmid but inserting a oligo with > the right sites. I face a very similar situation. I have a promoter/enhancer that is approximately 10 kb in size. The fragment I have begins approximately 1 kb before the farthest known protein binding site, where expression of the protein increases promoter activity rather dramatically (about 30-fold). The fragment ends about 100 bp after the known transcription start-site. We have accidentally discovered that there is an additional 600 bp _AFTER_ the transcription start-site that also increase promoter activity, most likely by participating in enhanceosome formation. There were no convenient restriction sites to use, and PCR of this fragment failed time & again. So, we ended up synthesizing the fragment as a "mini-gene"; we used a German company called Mr. Gene, but lots of places offer very competitive prices now. Because adding this fragment added a long 5'UTR, not to mention part of an ORF, we modified the reporter to include the EMCV IRES before the luciferase ORF. We decided to include the EMCV IRES as part of the "mini-gene" that was synthesized. The total length was about 1200 bp, and it cost us $600.00 + $50 in shipping charges. Synthesis took less than less than 4 weeks. I don't know how long your piece is, but you could consider having it synthesized as a "mini-gene". Ashok -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From rahimpour_a from yahoo.com Sat Jun 6 14:45:27 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Sat Jun 6 15:16:42 2009 Subject: oligo and generunner manual Message-ID: <368455.47237.qm@web52705.mail.re2.yahoo.com> Dear collegues dose any body has oligo5 or generunner manual? From prae from gmx.net Mon Jun 8 04:24:10 2009 From: prae from gmx.net (Christian Praetorius) Date: Mon Jun 8 13:42:54 2009 Subject: Promoter amplification References: <78nfj6F1mtj2uU1@mid.individual.net> <78pr1fF1krs6cU1@mid.individual.net> Message-ID: <7943lsF1p0fcrU1@mid.individual.net> Aawara Chowdhury wrote: >I don't know how long your piece is, but you could consider having >it synthesized as a "mini-gene". It's approximately 2,5kb, so my boss wouldn't be to glad about the costs for ordering such a large piece of DNA. Since the BAC has some appropriate restriction sites (I will only have to add them to the plasmid, which is quite simple), I will try to go this route. And keep the other one in my mind. Christian -- X-no-Sig: yes From prae from gmx.net Mon Jun 8 04:24:27 2009 From: prae from gmx.net (Christian Praetorius) Date: Mon Jun 8 13:43:04 2009 Subject: Promoter amplification References: <78nfj6F1mtj2uU1@mid.individual.net> <78pr1fF1krs6cU1@mid.individual.net> Message-ID: <7943mdF1p0fcrU2@mid.individual.net> dk@no.email.thankstospam.net (DK) wrote: >was: lower extension temperature to 65C with PfuFusion for >2 min/kb. Worked like a charm. Good to know. Christian -- X-no-Sig: yes From zineldeen from gmail.com Tue Jun 9 04:06:03 2009 From: zineldeen from gmail.com (doaa zineldeen) Date: Tue Jun 9 13:19:54 2009 Subject: Methods Digest, Vol 49, Issue 4 In-Reply-To: <200906041703.n54H3kp01451@net.bio.net> References: <200906041703.n54H3kp01451@net.bio.net> Message-ID: <440923e10906090206q2ec336eaqf183e5900fcb02aa@mail.gmail.com> Dear colleagues I would like to know if some one can help in this regard I am going to express a protein kinase as recombinant protein in SF9 cells the purified protein will be kinetically active is there possible way to make it kinetically inactive because I am going to use it as a substrate for in vitro kinase reaction if so does purification under denaturing condition help or adding specific kinase inhibitor in the last 4 h to the SF9 culture before harvesting Thank you in advance doaa From moh_aldeeb from yahoo.com Tue Jun 9 11:27:30 2009 From: moh_aldeeb from yahoo.com (M Aldeeb) Date: Tue Jun 9 13:20:02 2009 Subject: Total Protein (BioRad) Message-ID: <60860.39869.qm@web55808.mail.re3.yahoo.com> Dear All, I have a spectrophotometer (with cuvette). I want to measure total protein using BioRad Dye (BradFord method). How much protein (insect homogenate)and how much dye? Is it 100 uL Protein + 1900 uL (1X) Dye? @ 595 nm Please advise. Many thanks. Mohammad Ali Al-Deeb --- On Sat, 6/6/09, Azam Rahimpour wrote: > From: Azam Rahimpour > Subject: oligo and generunner manual > To: methods@magpie.bio.indiana.edu > Date: Saturday, June 6, 2009, 3:45 PM > Dear collegues > dose any body has oligo5 or generunner manual? > > > ? ? ? > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From moh_aldeeb from yahoo.com Tue Jun 9 05:12:24 2009 From: moh_aldeeb from yahoo.com (M Aldeeb) Date: Tue Jun 9 13:34:35 2009 Subject: Total Protein Message-ID: <429435.1867.qm@web55807.mail.re3.yahoo.com> Dear All, I have a spectrophotometer (with cuvette). I want to measure total protein using BioRad Dye (BradFord method). How much protein (insect homogenate)and how much dye? Is it 100 uL Protein + 1900 uL (1X) Dye? @ 595 nm Please advise. Many thanks. Mohammad Ali Al-Deeb From jinxwang from scau.edu.cn Wed Jun 10 05:06:38 2009 From: jinxwang from scau.edu.cn (Jinxiang Wang) Date: Wed Jun 10 10:47:54 2009 Subject: ER special dyes Message-ID: <444628504.28842@eyou.net> Dear Colleaguses, I am going to localize a protein which is from soybean.Bioinformatic analysis indicates it is maybe licalized in ER membrane.Initially I would likt to fuse my protein to GFP and check its localization.Alternatively I can employe antibody against my protein to verify its localization. Certainly the later technique is time-consuming. Would you kindly give me your suggestions related to special marker dyes for ER or ER-special marker proteins? In addtion I know some special dyes and antibody for ER-special marker proteins are commercially availabe. I look foward to comments or ideas of experts to guide me to select better dyes or marker protein Ab? Many thanks in advance. Best regards. Jinxiang -- ---------------------------------------------------- Jinxiang Wang Ph.D College of Resources and Environment Science Root Biology Center South China Agricultural University Guangzhou 510642, P.R.China Email: jinxwang@scau.edu.cn Tel: +86-20-85280156 (O) Fax: +86-20-85281829 From cathalgarvey from gmail.com Wed Jun 10 14:17:32 2009 From: cathalgarvey from gmail.com (Cathal Garvey) Date: Wed Jun 10 14:36:01 2009 Subject: ER special dyes In-Reply-To: <444628504.28842@eyou.net> References: <444628504.28842@eyou.net> Message-ID: <58FE978C-5C49-4506-8C2A-AED73F9EB867@gmail.com> I have it on good authority from a labmate that sucrose-gradients are a great way to check for protein localisation. I gather it involves a long centrifuge cycle but yields very reliable results. On 10 Jun 2009, at 11:06, Jinxiang Wang wrote: > Dear Colleaguses, > > I am going to localize a protein which is from soybean.Bioinformatic > analysis indicates it is maybe licalized in ER membrane.Initially I > would likt to fuse my protein to GFP and check its > localization.Alternatively I can employe antibody against my protein > to > verify its localization. Certainly the later technique is > time-consuming. Would you kindly give me your suggestions related to > special marker dyes for ER or ER-special marker proteins? In addtion I > know some special dyes and antibody for ER-special marker proteins are > commercially availabe. I look foward to comments or ideas of experts > to > guide me to select better dyes or marker protein Ab? Many thanks in > advance. > > Best regards. > > Jinxiang > > > -- > ---------------------------------------------------- > Jinxiang Wang Ph.D > > College of Resources and Environment Science > Root Biology Center > South China Agricultural University > Guangzhou 510642, P.R.China > > Email: jinxwang@scau.edu.cn > Tel: +86-20-85280156 (O) > Fax: +86-20-85281829 > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods From jinxwang from scau.edu.cn Fri Jun 12 06:14:19 2009 From: jinxwang from scau.edu.cn (Jinxiang Wang) Date: Fri Jun 12 07:30:57 2009 Subject: ER special dyes References: 444628504.28842@eyou.net Message-ID: <444805356.29602@eyou.net> -- ---------------------------------------------------- Jinxiang Wang Ph.D College of Resources and Environment Science Root Biology Center South China Agricultural University Guangzhou 510642, P.R.China Email: jinxwang@scau.edu.cn Tel: +86-20-85280156 (O) Fax: +86-20-85281829 From engelbert_buxbaum from hotmail.com Fri Jun 12 11:37:03 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri Jun 12 19:49:27 2009 Subject: ER special dyes References: Message-ID: Am 10.06.2009, 06:06 Uhr, schrieb Jinxiang Wang : > Dear Colleaguses, > > I am going to localize a protein which is from soybean.Bioinformatic > analysis indicates it is maybe licalized in ER membrane.Initially I > would likt to fuse my protein to GFP and check its > localization.Alternatively I can employe antibody against my protein to > verify its localization. Certainly the later technique is > time-consuming. Would you kindly give me your suggestions related to > special marker dyes for ER or ER-special marker proteins? In addtion I > know some special dyes and antibody for ER-special marker proteins are > commercially availabe. I look foward to comments or ideas of experts to > guide me to select better dyes or marker protein Ab? Many thanks in > advance. check Molecular Probes (Invitrogen.com) for ER-specicic life-cell markers. Alternatively of course you can use an fluorescent antibody against an ER-specific protein, however, then the cell has to be permeabilised. From salsag from essex.ac.uk Sat Jun 13 13:16:37 2009 From: salsag from essex.ac.uk (Alsagaby, Suliman) Date: Sat Jun 13 17:07:44 2009 Subject: Western Blotting problems Message-ID: Hello I did western blot twice and every time I have the same problem in which the PVDF membrane does not contain any protein although it contains protein marker. The bands were visualized by staining the gel. So it seems to me that the bands did not transfer to the membrane. I verified the transfer duration: one for 12h at 30V and the other one was for 1h at 100V. I am great full if any body can help me and direct me to the right protocol if I am doing wrong one. From Nikola.Wenta from nottingham.ac.uk Sun Jun 14 12:43:22 2009 From: Nikola.Wenta from nottingham.ac.uk (Nikola Wenta) Date: Sun Jun 14 20:09:35 2009 Subject: Western Blotting problems (Alsagaby, Suliman) In-Reply-To: <200906141703.n5EH3Jp02106@net.bio.net> References: <200906141703.n5EH3Jp02106@net.bio.net> Message-ID: <814A9FCB0AD790489679BFBCE6CA5E550273B75A@VUIEXCHC.ad.nottingham.ac.uk> Hi! As you are using a PVDF membran, did you wet the membrane with methanol? You didn't stain the gel with Coomassie before blotting, right? Are you sure that you really had protein on your gel (Ponceau)? Do you use semi-dry blot? Than you need to take care that you have your gel on top of the membrane. Niko > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu > [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of > methods-request@oat.bio.indiana.edu > Sent: Sonntag, 14. Juni 2009 18:03 > To: methods@magpie.bio.indiana.edu > Subject: Methods Digest, Vol 49, Issue 12 > > 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. Western Blotting problems (Alsagaby, Suliman) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Sat, 13 Jun 2009 19:16:37 +0100 > From: "Alsagaby, Suliman" > Subject: Western Blotting problems > To: > Message-ID: > > Content-Type: text/plain; charset="us-ascii" > > Hello > > I did western blot twice and every time I have the same > problem in which the PVDF membrane does not contain any > protein although it contains protein marker. The bands were > visualized by staining the gel. So it seems to me that the > bands did not transfer to the membrane. I verified the > transfer duration: one for 12h at 30V and the other one was > for 1h at 100V. > > I am great full if any body can help me and direct me to the > right protocol if I am doing wrong one. > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 49, Issue 12 > *************************************** > This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation. From stanleycheungkk from yahoo.com Sun Jun 14 10:43:33 2009 From: stanleycheungkk from yahoo.com (Stanley Cheung) Date: Sun Jun 14 20:09:44 2009 Subject: Western Blotting problems In-Reply-To: References: Message-ID: <407401.441.qm@web54507.mail.re2.yahoo.com> Hi Alsagaby, Some questions for you. 1. Do you have a positive control in your blot? 2. Are you sure your samples have the target protein in a detectable level? 3. Do you know if the quality of?antibodies (primary and secondary) is ok? 4. Anything is new in these blots, eg. sample buffers, transfer buffer, wash buffer, etc.? Sometimes, even if you see the protein marker transferred onto the membrane, it doesn't mean that the protein of your interest was well-resolved and transferred, because the protein marker is well-prepared and ready for running electrophoresis, while some proteins may require special conditions (urea, reducing agent,?up to 8%?SDS, etc). I heard that my colleaque uses the protein marker directly without adding sample buffer, but I like to use the same sample buffer as for?other samples on the same gel, otherwise, it may have strong distortion effect which I experienced many times before.?Moreover, I think?different?sample buffers should have effects on the mobility of the same protein. So, adding same sample buffer to all samples on the same gel should give a better estimation of the protein sizes. I don't know which way is correct. Any slightly change in your sample buffer may affect the solubility/denaturing of some tricky proteins, which means that you can't exclude the possibility that your target protein become insoluble or not well denatured when you are running the gel. So, the protein marker may not tell you enough information. Hope you will find out the problem very soon and let us know what it is. Cheers, Stanley ________________________________ From: "Alsagaby, Suliman" To: methods@magpie.bio.indiana.edu Sent: Sunday, June 14, 2009 2:16:37 AM Subject: Western Blotting problems Hello I did western blot twice and every time I have the same problem in which the PVDF membrane does not contain any protein although it contains protein marker. The bands were visualized by staining the gel. So it seems to me that the bands did not transfer to the membrane. I verified the transfer duration: one for 12h at 30V and the other one was for 1h at 100V. I am great full if any body can help me and direct me to the right protocol if I am doing wrong one. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods Send instant messages to your online friends http://uk.messenger.yahoo.com From TieQiao.Wu from bakeridi.edu.au Sun Jun 14 18:28:47 2009 From: TieQiao.Wu from bakeridi.edu.au (TieQiao Wu) Date: Sun Jun 14 20:09:52 2009 Subject: Methods Digest, Vol 49, Issue 12 In-Reply-To: <200906141703.n5EH3Jp02106@net.bio.net> References: <200906141703.n5EH3Jp02106@net.bio.net> Message-ID: <217613C88C00D4448B099AD7A374F253921D7B@exch01.bhri.internal> Hi, Did you use prestained protein marker? You should see the marker on your membrane very clearly using your transferring voltage and time setting. What I can suggest at the moment is that you check your transfer buffer and your sample buffer. Cheers -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of methods-request@oat.bio.indiana.edu Sent: Monday, 15 June 2009 03:03 AM To: methods@magpie.bio.indiana.edu Subject: Methods Digest, Vol 49, Issue 12 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. Western Blotting problems (Alsagaby, Suliman) ---------------------------------------------------------------------- Message: 1 Date: Sat, 13 Jun 2009 19:16:37 +0100 From: "Alsagaby, Suliman" Subject: Western Blotting problems To: Message-ID: Content-Type: text/plain; charset="us-ascii" Hello I did western blot twice and every time I have the same problem in which the PVDF membrane does not contain any protein although it contains protein marker. The bands were visualized by staining the gel. So it seems to me that the bands did not transfer to the membrane. I verified the transfer duration: one for 12h at 30V and the other one was for 1h at 100V. I am great full if any body can help me and direct me to the right protocol if I am doing wrong one. ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 49, Issue 12 *************************************** ______________________________________________________________________ This email has been scanned by the MessageLabs Email Security System. ______________________________________________________________________ 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 TieQiao.Wu from bakeridi.edu.au Sun Jun 14 18:28:47 2009 From: TieQiao.Wu from bakeridi.edu.au (TieQiao Wu) Date: Sun Jun 14 20:10:03 2009 Subject: Methods Digest, Vol 49, Issue 12 In-Reply-To: <200906141703.n5EH3Jp02106@net.bio.net> References: <200906141703.n5EH3Jp02106@net.bio.net> Message-ID: <217613C88C00D4448B099AD7A374F253921D7B@exch01.bhri.internal> Hi, Did you use prestained protein marker? You should see the marker on your membrane very clearly using your transferring voltage and time setting. What I can suggest at the moment is that you check your transfer buffer and your sample buffer. Cheers -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of methods-request@oat.bio.indiana.edu Sent: Monday, 15 June 2009 03:03 AM To: methods@magpie.bio.indiana.edu Subject: Methods Digest, Vol 49, Issue 12 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. Western Blotting problems (Alsagaby, Suliman) ---------------------------------------------------------------------- Message: 1 Date: Sat, 13 Jun 2009 19:16:37 +0100 From: "Alsagaby, Suliman" Subject: Western Blotting problems To: Message-ID: Content-Type: text/plain; charset="us-ascii" Hello I did western blot twice and every time I have the same problem in which the PVDF membrane does not contain any protein although it contains protein marker. The bands were visualized by staining the gel. So it seems to me that the bands did not transfer to the membrane. I verified the transfer duration: one for 12h at 30V and the other one was for 1h at 100V. I am great full if any body can help me and direct me to the right protocol if I am doing wrong one. ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 49, Issue 12 *************************************** ______________________________________________________________________ This email has been scanned by the MessageLabs Email Security System. ______________________________________________________________________ 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 amanosz from yahoo.com Tue Jun 16 08:09:18 2009 From: amanosz from yahoo.com (David Liu) Date: Tue Jun 16 10:18:57 2009 Subject: How to exchange antibiotic resistance markers on plasmids for E coli Message-ID: <728326.41571.qm@web36506.mail.mud.yahoo.com> Hello, Has anyone exchanged or heard of someone exchanging the antibiotic resistance marker on a plasmid for E. coli? I need to co-transform E coli with two plasmids that have the same resistance marker, and the plasmids are not available with any other marker. I have plasmid sequence information for the two plasmids of interest and a 3rd plasmid with the new resistance marker that identifies the location of each marker. I've confirmed with a BLAST search that the annotated sequences are simply the coding sequences for each marker (start to stop codon). Is it simply a modular procedure where I remove one ORF and replace it with the other? Or are there other considerations I need to think about? The plasmid map doesn't annotate any promoter or terminator sequence for the resistance marker. But I assume if one is necessary, then I am only substituting the coding sequence and the remaining un-annotated architecture is still present. For what it's worth, I'd like to substitute a chloramphenicol resistance marker with a kanamycin marker. Thanks in advance for your help, David From fulvio.celsi from gmail.com Tue Jun 16 10:36:00 2009 From: fulvio.celsi from gmail.com (Fulvio Celsi) Date: Tue Jun 16 12:57:30 2009 Subject: How to exchange antibiotic resistance markers on plasmids for E coli In-Reply-To: <728326.41571.qm@web36506.mail.mud.yahoo.com> References: <728326.41571.qm@web36506.mail.mud.yahoo.com> Message-ID: Dear David why not simply move the gene instead of the resistance? easier.... Fulvio Celsi BsC,PhD Sector of Neurobiology, International School for Advanced Studies, Scuola Internazionale di Studi Superiori Avanzati (SISSA), Trieste Italy Mobile: +393286489131 2009/6/16 David Liu > > Hello, > > Has anyone exchanged or heard of someone exchanging the antibiotic > resistance marker on a plasmid for E. coli? I need to co-transform E coli > with two plasmids that have the same resistance marker, and the plasmids are > not available with any other marker. I have plasmid sequence information > for the two plasmids of interest and a 3rd plasmid with the new resistance > marker that identifies the location of each marker. I've confirmed with a > BLAST search that the annotated sequences are simply the coding sequences > for each marker (start to stop codon). Is it simply a modular procedure > where I remove one ORF and replace it with the other? Or are there other > considerations I need to think about? > > The plasmid map doesn't annotate any promoter or terminator sequence for > the resistance marker. But I assume if one is necessary, then I am only > substituting the coding sequence and the remaining un-annotated architecture > is still present. > > For what it's worth, I'd like to substitute a chloramphenicol resistance > marker with a kanamycin marker. > > Thanks in advance for your help, > > David > > > > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From gerchman from research.haifa.ac.il Tue Jun 16 13:00:00 2009 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Tue Jun 16 15:32:32 2009 Subject: How to exchange antibiotic resistance markers on plasmids for E. coli Message-ID: <1245175200.4a37dda038b92@webmail.haifa.ac.il> Unfortunately it is not that simple. To co-transform E. coli, and have stable transformation you have to have: 1. Two different selection markers on the plasmids (e.g. resistance genes) AND 2. Two different origin of replication markers on the plasmids- e.g. pA15 (from pACYC184) with ColE1 (pBR322) or another combination. Mind you, pUC19 origine IS NOT compatible with ColE1 as they basicaly the same origin. Given this you might be better off transfering your part of interest from your current plasmid to a more compatible plasmid. This will probably also take care of the resistance gene issue. You can more on this 'origin of replication' compatibility issue in this web-page: Specifically to you question, for the promoter site just take 100~200 bp upstream of the first ATG and you are covered. Hope this help. Yoram gerchman AT research DOT haifa DOT ac DOT il Quoting methods-request@oat.bio.indiana.edu: > > Date: Tue, 16 Jun 2009 06:09:18 -0700 (PDT) > From: David Liu > Subject: How to exchange antibiotic resistance markers on plasmids for > E coli > To: methods@magpie.bio.indiana.edu > Message-ID: <728326.41571.qm@web36506.mail.mud.yahoo.com> > Content-Type: text/plain; charset=us-ascii > > > Hello, > > Has anyone exchanged or heard of someone exchanging the antibiotic resistance > marker on a plasmid for E. coli? I need to co-transform E coli with two > plasmids that have the same resistance marker, and the plasmids are not > available with any other marker. I have plasmid sequence information for the > two plasmids of interest and a 3rd plasmid with the new resistance marker > that identifies the location of each marker. I've confirmed with a BLAST > search that the annotated sequences are simply the coding sequences for each > marker (start to stop codon). Is it simply a modular procedure where I > remove one ORF and replace it with the other? Or are there other > considerations I need to think about? > > The plasmid map doesn't annotate any promoter or terminator sequence for the > resistance marker. But I assume if one is necessary, then I am only > substituting the coding sequence and the remaining un-annotated architecture > is still present. > > For what it's worth, I'd like to substitute a chloramphenicol resistance > marker with a kanamycin marker. > > Thanks in advance for your help, > > David > ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From amanosz from yahoo.com Wed Jun 17 10:38:30 2009 From: amanosz from yahoo.com (David Liu) Date: Wed Jun 17 11:57:56 2009 Subject: How to exchange antibiotic resistance markers on plasmids for E. coli Message-ID: <137315.74776.qm@web36503.mail.mud.yahoo.com> Actually, just started reading about the issue of origin of replications as well, and will end up moving the gene to a different plasmid with separate marker and ori, so original problem is solved. Thanks! But for the sake of discussion, wouldn't it be more ideal to have both different resistance marker *and* origin? Sure it sounds doable to just have different resistance marker, but wouldn't asymmetric plasmid doubling potentially dilute one plasmid to effectively low copy number? For example, two plasmids sharing an ori with copy number, say 40. If you're unlucky at picking clones, you could conceivably result in a cell with 39 copies of one plasmid and 1 copy of the other. Both resistance markers are present to satisfy the selection requirements but you'd have uneven protein expression, promoter differences notwithstanding. David --- On Tue, 6/16/09, DK wrote: > From: DK > Subject: Re: How to exchange antibiotic resistance markers on plasmids for E. coli > To: methods@magpie.bio.indiana.edu > Date: Tuesday, June 16, 2009, 4:51 PM > In article , > Yoram Gerchman > wrote: > > > > > >Unfortunately it is not that simple. > > It is. E.coli does not have an active mechanism of plasmid > > incompatibility. The loss of one plasmid in the case of > two plasmids carrying the same ori and selection marker > is simply a dilution effect. > > >To co-transform E. coli, and have stable transformation > you have to have: > >1. Two different selection markers on the plasmids > (e.g. resistance genes) > >AND > >2. Two different origin of replication markers on the > plasmids- e.g. pA15 (from > >pACYC184) with ColE1 (pBR322) or another combination. > Mind you, pUC19 origine > > IS > >NOT compatible with ColE1 as they basicaly the same > origin. > > It's *either* 1) or 2) above, not *and*! > > We routinely co-transform pET31 and pET24, which are > essentially > the same plasmids carrying different resistance. Stable > enough to > get a large colony, grow an overnight culture and expand > into 12 liters > of culture - without losing expression of both of the genes > cloned > into the plasmids. > > DK > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From gerchman from research.haifa.ac.il Thu Jun 18 09:40:21 2009 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Thu Jun 18 10:03:01 2009 Subject: How to exchange antibiotic resistance markers on plasmids for E. coli In-Reply-To: <200906171704.n5HH4ap15952@net.bio.net> References: <200906171704.n5HH4ap15952@net.bio.net> Message-ID: <1245336021.4a3a51d544ede@webmail.haifa.ac.il> DK and David Indeed it is the dilution factor, not an active mechanism (if we ignore the copy control). I must say though I tried same origin + different markers and vise verse combinations in the past and many times ended up loosing one of the plasmid (not always, but enough). It really is a question of how lucky you feel, if you use BOTH different origin AND different markers you are on the safe side. Just my 2 cents Yoram > Date: Tue, 16 Jun 2009 23:51:19 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: How to exchange antibiotic resistance markers on plasmids > > In article , Yoram Gerchman > wrote: > > > >Unfortunately it is not that simple. > > It is. E.coli does not have an active mechanism of plasmid > incompatibility. The loss of one plasmid in the case of > two plasmids carrying the same ori and selection marker > is simply a dilution effect. > > >To co-transform E. coli, and have stable transformation you have to have: > >1. Two different selection markers on the plasmids (e.g. resistance genes) > >AND > >2. Two different origin of replication markers on the plasmids- e.g. pA15 > (from > >pACYC184) with ColE1 (pBR322) or another combination. Mind you, pUC19 > origine > > IS > >NOT compatible with ColE1 as they basicaly the same origin. > > It's *either* 1) or 2) above, not *and*! > > We routinely co-transform pET31 and pET24, which are essentially > the same plasmids carrying different resistance. Stable enough to > get a large colony, grow an overnight culture and expand into 12 liters > of culture - without losing expression of both of the genes cloned > into the plasmids. > > DK > > But for the sake of discussion, wouldn't it be more ideal to have both > different resistance marker *and* origin? Sure it sounds doable to just have > different resistance marker, but wouldn't asymmetric plasmid doubling > potentially dilute one plasmid to effectively low copy number? For example, > two plasmids sharing an ori with copy number, say 40. If you're unlucky at > picking clones, you could conceivably result in a cell with 39 copies of one > plasmid and 1 copy of the other. Both resistance markers are present to > satisfy the selection requirements but you'd have uneven protein expression, > promoter differences notwithstanding. > > David > > --- On Tue, 6/16/09, DK wrote: > > > From: DK > > Subject: Re: How to exchange antibiotic resistance markers on plasmids for > E. coli > > To: methods@magpie.bio.indiana.edu > > Date: Tuesday, June 16, 2009, 4:51 PM > > In article , > > Yoram Gerchman > > wrote: > > > > > > > > >Unfortunately it is not that simple. > > > > It is. E.coli does not have an active mechanism of plasmid > > > > incompatibility. The loss of one plasmid in the case of > > two plasmids carrying the same ori and selection marker > > is simply a dilution effect. > > > > >To co-transform E. coli, and have stable transformation > > you have to have: > > >1. Two different selection markers on the plasmids > > (e.g. resistance genes) > > >AND > > >2. Two different origin of replication markers on the > > plasmids- e.g. pA15 (from > > >pACYC184) with ColE1 (pBR322) or another combination. > > Mind you, pUC19 origine > > > IS > > >NOT compatible with ColE1 as they basicaly the same > > origin. > > > > It's *either* 1) or 2) above, not *and*! > > > > We routinely co-transform pET31 and pET24, which are > > essentially > > the same plasmids carrying different resistance. Stable enough > > to get a large colony, grow an overnight culture and expand > > into 12 liters of culture - without losing expression of both > > of the genes cloned into the plasmids. > > > > DK > ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From tonightsthenight from gmail.com Tue Jun 23 13:43:54 2009 From: tonightsthenight from gmail.com (hippie dream) Date: Tue Jun 23 13:48:03 2009 Subject: Lectins Message-ID: <24170413.post@talk.nabble.com> Hello All, I recently purchased some TRITC conjugated Lectin from Triticum vulgaris from Sigma (L5266-2MG). I intend to use this lectin to label EPS components in a river biofilm. The lectin is shipped as a lyophilized powder. I following the instruction of Neu & Lawrence (Methods in Microbiology, 2004). They suggest preparing a stock solution by adding 1mL of buffer for every 1mg of lectin. My issue is that they suggest using either filter sterilized water or a buffer. My quandary is that I don't know which to use. I don't even really know how to assess which one to use and if I am using a buffer other than water which do I use? If anyone has any ideas that would be fantastic. Thanks! Sam -- View this message in context: http://www.nabble.com/Lectins-tp24170413p24170413.html Sent from the Bio.net - Methods mailing list archive at Nabble.com. From nick.theodorakis from gmail.com Tue Jun 23 14:55:26 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Tue Jun 23 23:17:41 2009 Subject: Lectins References: Message-ID: On Jun 23, 2:43?pm, hippie dream wrote: > Hello All, > > I recently purchased some TRITC conjugated Lectin from Triticum vulgaris > from Sigma (L5266-2MG). I intend to use this lectin to label EPS components > in a river biofilm. The lectin is shipped as a ?lyophilized powder. I > following the instruction of Neu & Lawrence (Methods in Microbiology, 2004). > They suggest preparing a stock solution by adding 1mL of buffer for every > 1mg of lectin. > > My issue is that they suggest using either filter sterilized water or a > buffer. My quandary is that I don't know which to use. I don't even really > know how to assess which one to use and if I am using a buffer other than > water which do I use? If anyone has any ideas that would be fantastic. > Check the product sheet that came with the sample. If it was lyophilized from buffer, then all you need to add is water because buffer should be reconstituted along with the lectin. Otherwise, choose conditions that are most compatibile with the solubility of the lectin and won't interfere with downstream applications. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From biotech.kunal13 from gmail.com Wed Jun 24 03:27:00 2009 From: biotech.kunal13 from gmail.com (KUNAL KALE) Date: Wed Jun 24 08:02:16 2009 Subject: help in PCR Message-ID: resp.sir, I am studant of PhD. in S.G.B. Amravati university , (India). and I am Working on Chloroplast Gene. I am working on an amplification of a gene that is 1369 bp. I have problem in amplification. With this mail I m sending attachment of sample of result which obtain after PCR . primers have been checked and seem to be working fine . I am Cerrently Using biometra gradient thermo cycler . I am cheking other primer of mec A gene which give Proper result .pleas give me suggetion on this problem.Your valuble suggetion Definatly help me in my research . If anyone has experience amplifying fragments this large I would love to discuss the methods by which they are using. your sincerly Mr. kunal kale From tonightsthenight from gmail.com Tue Jun 23 23:29:07 2009 From: tonightsthenight from gmail.com (hippie dream) Date: Wed Jun 24 08:02:23 2009 Subject: Lectins In-Reply-To: References: <24170413.post@talk.nabble.com> Message-ID: <24178504.post@talk.nabble.com> On Jun 23, 2:43?pm, hippie dream wrote: > Hello All, > > I recently purchased some TRITC conjugated Lectin from Triticum vulgaris > from Sigma (L5266-2MG). I intend to use this lectin to label EPS > components > in a river biofilm. The lectin is shipped as a ?lyophilized powder. I > following the instruction of Neu & Lawrence (Methods in Microbiology, > 2004). > They suggest preparing a stock solution by adding 1mL of buffer for every > 1mg of lectin. > > My issue is that they suggest using either filter sterilized water or a > buffer. My quandary is that I don't know which to use. I don't even really > know how to assess which one to use and if I am using a buffer other than > water which do I use? If anyone has any ideas that would be fantastic. > >Check the product sheet that came with the sample. If it was lyophilized from buffer, then all you need to add is water because buffer should be reconstituted along with the lectin. Thanks. Yeah it was definitely lyophilized from the buffer so all I had to do was add water. Sorry for the simple question. Thanks alot for the help. Sam -- View this message in context: http://www.nabble.com/Lectins-tp24170413p24178504.html Sent from the Bio.net - Methods mailing list archive at Nabble.com. From R.Jayakumar from roswellpark.org Wed Jun 24 12:09:30 2009 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Wed Jun 24 12:13:52 2009 Subject: help in PCR In-Reply-To: References: Message-ID: We do routine amplification of large sized genes from human genome. Your attachment did not come through, since bionet does not accept attachments. You could describe the problem in your email and we can try to help you. Jay ________________________________________ From: methods-bounces@oat.bio.indiana.edu [methods-bounces@oat.bio.indiana.edu] On Behalf Of KUNAL KALE [biotech.kunal13@gmail.com] Sent: Wednesday, June 24, 2009 4:27 AM To: methods@iubio.bio.indiana.edu Subject: help in PCR resp.sir, I am studant of PhD. in S.G.B. Amravati university , (India). and I am Working on Chloroplast Gene. I am working on an amplification of a gene that is 1369 bp. I have problem in amplification. With this mail I m sending attachment of sample of result which obtain after PCR . primers have been checked and seem to be working fine . I am Cerrently Using biometra gradient thermo cycler . I am cheking other primer of mec A gene which give Proper result .pleas give me suggetion on this problem.Your valuble suggetion Definatly help me in my research . If anyone has experience amplifying fragments this large I would love to discuss the methods by which they are using. your sincerly Mr. kunal kale 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 rijutak from googlemail.com Wed Jun 24 12:46:23 2009 From: rijutak from googlemail.com (Rijuta Garapaty) Date: Wed Jun 24 16:26:26 2009 Subject: help in PCR Message-ID: <71d8f0270906241046v4272b4ahc712f34ce23bf48c@mail.gmail.com> > From: KUNAL KALE > Subject: help in PCR > To: "methods@iubio.bio.indiana.edu" > Message-ID: > > Content-Type: text/plain; charset="iso-8859-1" > > resp.sir, > I am studant of PhD. in S.G.B. Amravati university , (India). and I am > Working on Chloroplast Gene. I am working on an amplification of a gene > that > is 1369 bp. I have problem in amplification. With this mail I m sending > attachment of sample of result which obtain after PCR . primers have been > checked and seem to be working fine . I am Cerrently Using biometra > gradient thermo cycler . I am cheking other primer of mec A gene which give > Proper result .pleas give me suggetion on this problem.Your valuble > suggetion Definatly help me in my research . > If anyone has experience amplifying fragments this large I would love to > discuss the methods by which they are using. > > > your sincerly > > Mr. kunal kale > > ------------------------------ > Did you vary other PCR conditions, like the primer annealing temperature or the number of cycles? Did you add additives like BSA to check whether your desired product is formed? Also, did you try varying the primer concentration in your PCR mix? While amplifying such a big fragment, the choice of polymerase is also very crucial. Rijuta From pjie2 from cam.ac.uk Wed Jun 24 18:55:08 2009 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Wed Jun 24 20:19:43 2009 Subject: help in PCR In-Reply-To: References: Message-ID: <7afsnkF1vfkkcU1@mid.individual.net> Rijuta Garapaty wrote: > > Did you vary other PCR conditions, like the primer annealing temperature or > the number of cycles? Did you add additives like BSA to check whether your > desired product is formed? Also, did you try varying the primer > concentration in your PCR mix? While amplifying such a big fragment, the > choice of polymerase is also very crucial. Since when was 1.4k a big fragment? That should be doable with any polymerase on the market under standard conditions. Check your primer sequence and concentration, and your DNA quality / concentration. Peter From sakkthi18 from gmail.com Wed Jun 24 22:14:44 2009 From: sakkthi18 from gmail.com (Sakkthi subramaniyam) Date: Wed Jun 24 23:20:15 2009 Subject: Acid phosphatase Message-ID: Dear sir / madam, Could any one tell how much concentration of acid phosphatase will be found in ripened and unripened fruits in mg/g. If you can give the concentrations for grapes it will be more useful. Thanks. I searched through in internet and books but couldn't get exact results. -- With regards, Sakkthi subramaniyam, From pattisyang from gmail.com Thu Jun 25 05:01:21 2009 From: pattisyang from gmail.com (Xuan Yang) Date: Thu Jun 25 08:18:20 2009 Subject: Puzzling ICP-ES Results, Metal Binding Protein? Message-ID: Hi, there. ICP-ES test was performed using my protein which was expressed and purified from E.coli strain Origami(DE3), in order to find out whether it bond metal or not. The results were puzzling. Without His tag, the protein concentration was ~34 micromolar, while the Zn was about ~17 micromolar, Fe was about ~8 micromolar, Cu was ~0.3 micromolar, Ni was ~0.4 micromolar. Other metals were negligible including Co, Cr, Mg. Buffer were used as references. In a separate test, the protein with His tag was ~47 micromolar, while Zn was ~24 micromolar, Fe was ~9 micromolar, Cu was ~6 micromolar, Ni was ~4 micromolar. Other metals were negligible including Co, Cr, Mg. Buffer were used as references. Well, based on these results, I suspected my protein was a zinc binding protein. However, I could not rule out other possibilities, especially Fe. Have anyone performed ICP-ES before? Is it a reliable method? Could a Zinc binding protein also bind Iron? Are there any method available, other than in-cell NMR, to determine whether my protein bind metal or not in vivo? Thanks in advance! Sincerely, Pattis >From IBP, CAS, Beijing Corr: pattisyang@gmail.com From rijutak from googlemail.com Thu Jun 25 12:21:18 2009 From: rijutak from googlemail.com (Rijuta Garapaty) Date: Thu Jun 25 14:32:19 2009 Subject: Methods Digest, Vol 49, Issue 18 In-Reply-To: <200906251703.n5PH3vp12751@net.bio.net> References: <200906251703.n5PH3vp12751@net.bio.net> Message-ID: <71d8f0270906251021u50ed3c7g6457f511e59f8039@mail.gmail.com> > > > Message: 4 > Date: Thu, 25 Jun 2009 02:42:34 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: help in PCR > To: methods@net.bio.net > Message-ID: > > In article <7afsnkF1vfkkcU1@mid.individual.net>, Peter Ellis < > pjie2@cam.ac.uk> wrote: > >Rijuta Garapaty wrote: > >> > >> Did you vary other PCR conditions, like the primer annealing temperature > or > >> the number of cycles? Did you add additives like BSA to check whether > your > >> desired product is formed? Also, did you try varying the primer > >> concentration in your PCR mix? While amplifying such a big fragment, the > >> choice of polymerase is also very crucial. > > > >Since when was 1.4k a big fragment? > > It was. About 25 years ago. At least, I remember talks of "long PCR", > meaning 3 kbp, then. :-) > > I am extremely sorry about that comment, instead of reading it as 1369 bp, i read it as 13690 bp :(. As Peter said, 1.4 kbp fragment can very easily be amplified using any polymerase under the standard conditions. I was definitely wrong! From editor from gene-quantification.info Fri Jun 26 03:21:08 2009 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Fri Jun 26 12:06:45 2009 Subject: qPCR NEWS June 2009 - MIQE media & press review Message-ID: qPCR NEWS June 2009 - MIQE media & press review --------------------------------------------------------------------- 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: - MIQE - media & press review - For better navigation we developed a TAG CLOUD =3D> http://directory.gene-quantification.info/ - NEW qPCR / real-time PCR BLOG =3D> http://real-time-pcr.blogspot.com/ - New qPCR events in autumn 2009 - New qPCR workshop modules at the TATAA Biocenter Germany =3D> http://tataa.gene-quantification.info/ European wide qPCR application workshops =3D> register now ! =3D> course program spring - summer - autumn 2009 =3D> http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pd= f =3D> http://www.gene-quantification.de/bioeps-courses-autumn-2009.pdf ---------------------------------------------------------------------------= ----- MIQE - media & press review =3D> http://www.gene-quantification.de/miqe.html#press International Scientists Secure Quality in Molecular Diagnostics Consensus Guideline Reached For Quantitative Polymerase Chain Reaction Press release by TATAA Biocenter Internationale Wissenschaftler sorgen f=FCr Qualit=E4tssicherung in der Molekulardiagnostik Real-timePCR data markup language Letter of the MIQE authors Letter to leading journals recommending the use of MIQE for quality control of qPCR experiments. IBT of the Academy of Sciences of the Czech Republic qPCR Grows Up by genome web qPCR Assay Quality assessment on SciTopics Quest Agrees to Pay Fine for Misbranding Tests Advancing DNA research safely and securely What do you hope to achieve with the guidelines? Data that Meets the MIQE Guidelines on Canadian BioTechnologist 2.0 GLOSSARY OF REAL-TIME PCR TERMS by M.Tevfik Dorak ---------------------------------------------------------------------------= ----- The MIQE Guidelines Minimum Information for Publication of Quantitative Real-Time PCR Experiments Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson, Jan Hellemans, Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W. Pfaffl, Gregory L. Shipley, Jo Vandesompele, & Carl T. Wittwer Clinical Chemistry 2009, 55(4): 611-622 =3D> http://miqe.gene-quantification.info =3D> http://www.gene-quantification.de/miqe-bustin-et-al-clin-chem-2009.pdf RDML: structured language and reporting guidelines for real-time quantitative PCR data Lefever S, Hellemans J, Pattyn F, Przybylski DR, Taylor C, Geurts R, Untergasser A, Vandesompele J; on behalf of the RDML consortium. Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium. Nucleic Acids Res. 2009 Apr;37(7): 2065-2069 =3D> http://rdml.gene-quantification.info =3D> http://www.gene-quantification.de/lefever-et-al-rdml-2009.pdf Reliability of real-time reverse-transcription PCR in clinical diagnostics: gold standard or substandard? Murphy J, Bustin SA. Expert Rev Mol Diagn. 2009 9(2):187-197 =3D> http://www.gene-quantification.de/murphy-bustin-review-qpcr-optimizati= on-2009.pdf Unreliable real-time PCR analysis of human endogenous retrovirus-W (HERV-W) RNA expression and DNA copy number in multiple sclerosis. Garson JA, Huggett JF, Bustin SA, Pfaffl MW, Benes V, Vandesompele J, Shipley GL. AIDS Res Hum Retroviruses. 2009 25(3): 377-378 =3D> http://www.gene-quantification.de/garson-et-al-aids-research-2009.pdf Real-time polymerasechain reaction =96 towardsa more reliable, accurateand relevant assay. SA Bustin EUROPEAN PHARMACEUTICAL REVIEW 2008 (6): 19-27 =3D> http://www.gene-quantification.de/bustin-review-qpcr-optimization-2009= .pdf In-House Nucleic Acid Amplification Assays in Research: How Much Quality ControlIs Needed before One Can Rely upon the Results? Petra Apfalter, UdoReischl and Margaret R. 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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 - 2009 All rights reserved. Any unauthorized use, reproduction, or transfer of this message or its contents, in any medium, is strictly prohibited. Disclaimer & Copyrights are displayed on the homepage www.gene-quantification.com To subscribe or change your e-mail address in qPCR NEWS, and if you would like to receive future issues FREE of charge, please send an e- mail with the subject SUBSCRIBE to mailto:newsletter@gene- quantification.info?subject=3DSUBSCRIBE From mccraight from biddulph.staffs.sch.uk Fri Jun 26 05:23:10 2009 From: mccraight from biddulph.staffs.sch.uk (mccraight) Date: Fri Jun 26 12:07:07 2009 Subject: agarose electrophoresis tank @ $10 Message-ID: <000501c9f648$1af48340$5d0d860a@BIDDULPH.INTERNAL> Hi, I am a Lab technician in a school,and we need an electophoresis tank to enable our 6th for students to follow a new forensic science course. Money in school is very limited,so I am hoping to make one. Would it be possible for you to send me details on how to construct a simple tank,and whether ordinary agar could be used instead of agarose as this would also cut costs. Kind Regards Steph McCraight From ariajohnson from gmail.com Fri Jun 26 12:21:54 2009 From: ariajohnson from gmail.com (aria johnson) Date: Fri Jun 26 12:53:20 2009 Subject: agarose electrophoresis tank @ $10 In-Reply-To: <000501c9f648$1af48340$5d0d860a@BIDDULPH.INTERNAL> References: <000501c9f648$1af48340$5d0d860a@BIDDULPH.INTERNAL> Message-ID: Hi Check this website out for simple tank http://www.science-projects.com/GE/HorizChamber.htm not sure whether agar will give you the resolution you need for the exercise. Will you use EtBr and UV for visualization? Im not sure what age is 6th form but you may want to monitor the EtBr step or find an alternate way to visualize your pcr product. take care aj On Fri, Jun 26, 2009 at 6:23 AM, mccraight wrote: > Hi, I am a Lab technician in a school,and we need an electophoresis tank to > enable our 6th for students to follow a new forensic science course. Money > in school is very limited,so I am hoping to make one. Would it be possible > for you to send me details on how to construct a simple tank,and whether > ordinary agar could be used instead of agarose as this would also cut costs. > > Kind Regards > > Steph McCraight > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- 'Being Happy doesn't mean everything's perfect--- It just means you've decided to see beyond the imperfections' From taliaferroD from mail.nih.gov Fri Jun 26 13:47:55 2009 From: taliaferroD from mail.nih.gov (Dwayne Taliaferro) Date: Fri Jun 26 15:22:47 2009 Subject: agarose electrophoresis tank @ $10 In-Reply-To: Message-ID: You could send an email and/or visit your local college or university's chemistry or biology department and see if someone will give or let you borrow one. I'm willing to bet that they would be willing to donate all the reagents. On 6/26/09 1:21 PM, "aria johnson" wrote: > Hi Check this website out for simple tank > http://www.science-projects.com/GE/HorizChamber.htm > not sure whether agar will give you the resolution you need for the > exercise. > Will you use EtBr and UV for visualization? Im not sure what age is 6th form > but you may want to monitor the EtBr step or find an alternate way > to visualize your pcr product. > take care > aj > > On Fri, Jun 26, 2009 at 6:23 AM, mccraight > wrote: > >> Hi, I am a Lab technician in a school,and we need an electophoresis tank to >> enable our 6th for students to follow a new forensic science course. Money >> in school is very limited,so I am hoping to make one. Would it be possible >> for you to send me details on how to construct a simple tank,and whether >> ordinary agar could be used instead of agarose as this would also cut costs. >> >> Kind Regards >> >> Steph McCraight >> _______________________________________________ >> Methods mailing list >> Methods@net.bio.net >> http://www.bio.net/biomail/listinfo/methods >> > > From engelbert_buxbaum from hotmail.com Fri Jun 26 15:13:51 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri Jun 26 15:22:54 2009 Subject: Lectins References: <24170413.post@talk.nabble.com> Message-ID: Am 24.06.2009, 00:29 Uhr, schrieb hippie dream : >> Check the product sheet that came with the sample. If it was > lyophilized from buffer, then all you need to add is water because > buffer should be reconstituted along with the lectin. > > > Thanks. Yeah it was definitely lyophilized from the buffer so all I had > to > do was add water. Sorry for the simple question. Thanks alot for the > help. If you plan longer-term storage of the stock solution and the product specifications do not state otherwise, consider adding an equal amount of glycerol to the solution and store at -20 celsius. Because of the glycerol the solution will not freeze, avoiding damage by freeze-thaw cycles. From engelbert_buxbaum from hotmail.com Fri Jun 26 15:21:21 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri Jun 26 16:13:38 2009 Subject: agarose electrophoresis tank @ $10 References: <000501c9f648$1af48340$5d0d860a@BIDDULPH.INTERNAL> Message-ID: Am 26.06.2009, 13:21 Uhr, schrieb aria johnson : > Hi Check this website out for simple tank > http://www.science-projects.com/GE/HorizChamber.htm > not sure whether agar will give you the resolution you need for the > exercise. > Will you use EtBr and UV for visualization? Im not sure what age is 6th > form > but you may want to monitor the EtBr step or find an alternate way > to visualize your pcr product. > take care > aj > > On Fri, Jun 26, 2009 at 6:23 AM, mccraight > > wrote: > >> Hi, I am a Lab technician in a school,and we need an electophoresis >> tank to >> enable our 6th for students to follow a new forensic science course. >> Money >> in school is very limited,so I am hoping to make one. Would it be >> possible >> for you to send me details on how to construct a simple tank,and whether >> ordinary agar could be used instead of agarose as this would also cut >> costs. Agarose for electrophoresis has been purified to contain few if any charged groups, which would cause a phenomenon called electroendosmosis. This can seriously degrade resolution. Exception: Counter-stream immunoelectrophoresis and certain modes of capillary electrophoresis, where electroendosmosis is actually required for separation. From tonightsthenight from gmail.com Fri Jun 26 16:00:36 2009 From: tonightsthenight from gmail.com (Sam Albers) Date: Fri Jun 26 16:13:46 2009 Subject: Lectins In-Reply-To: References: <24170413.post@talk.nabble.com> Message-ID: > > Check the product sheet that came with the sample. If it was >>> >> lyophilized from buffer, then all you need to add is water because >> buffer should be reconstituted along with the lectin. >> >> >> Thanks. Yeah it was definitely lyophilized from the buffer so all I had to >> do was add water. Sorry for the simple question. Thanks alot for the help. >> > > If you plan longer-term storage of the stock solution and the product > specifications do not state otherwise, consider adding an equal amount of > glycerol to the solution and store at -20 celsius. Because of the glycerol > the solution will not freeze, avoiding damage by freeze-thaw cycles. > > Thanks for the suggestion. I think that storing the stock solution at -20 will be an issue like you said but since I am going to be using the lectin for only a couple months I think it will be fine to just store it at 4 degrees. From gerchman from research.haifa.ac.il Sat Jun 27 14:28:43 2009 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Sat Jun 27 17:07:52 2009 Subject: agarose electrophoresis tank @ $10 (aria johnson) Message-ID: <1246130923.4a4672eb427d2@webmail.haifa.ac.il> Try this webpage: Very simple and cheap apparatus, they even switched EtBr with Methylen Blue! so no carcinogenic compounds and no UV trans-illuminator needed. Nice A few suggestions though: 1. If you can use silver wire rather then stainless (you can even use iron wire/nails but they will build turf with time. 2. If you can afford it get some Agarose rather then Agar Agar, the results will be cleaner. You can recycle the agarose for a while by re-boiling it. You will get some background but usually its not that bad (for a while). You can pour your gel directly in the running box, just place a piece of Styrofoam on each side, pull it out when the gel solidified, and fill with buffer. Then put in your electrodes. Cheap PCR recipe here: And some more gel ideas here An PDF of one of the Scientific American "Amateur Scientist" wonderful column Hope this helps. Yoram gerchman AT research DOT haifa DOT ac DOT il ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From cathalgarvey from gmail.com Mon Jun 29 03:56:27 2009 From: cathalgarvey from gmail.com (Cathal Garvey) Date: Mon Jun 29 14:11:47 2009 Subject: agarose electrophoresis tank @ $10 In-Reply-To: References: <000501c9f648$1af48340$5d0d860a@BIDDULPH.INTERNAL> Message-ID: <468b1a400906290156u14a114a2r9d3b0c982d5a8c75@mail.gmail.com> You might enjoy this article in the Science Creative Quarterly, which explains how to use available-at-supermarket ingredients to perform DNA extractions, gel electrophoresis and staining. http://is.gd/1hPJs The short answer is: you can use agar, though the band will be a little blurry, and you can use a blue food colouring to stain the DNA, the specific name is in the article. Your agar might need to be good and pure, but if it's just to demonstrate the principal of electrophoresis it's fine. 2009/6/26 Dr Engelbert Buxbaum > Am 26.06.2009, 13:21 Uhr, schrieb aria johnson : > > Hi Check this website out for simple tank >> http://www.science-projects.com/GE/HorizChamber.htm >> not sure whether agar will give you the resolution you need for the >> exercise. >> Will you use EtBr and UV for visualization? Im not sure what age is 6th >> form >> but you may want to monitor the EtBr step or find an alternate way >> to visualize your pcr product. >> take care >> aj >> >> On Fri, Jun 26, 2009 at 6:23 AM, mccraight < >> mccraight@biddulph.staffs.sch.uk >> >>> wrote: >>> >> >> Hi, I am a Lab technician in a school,and we need an electophoresis tank >>> to >>> enable our 6th for students to follow a new forensic science course. >>> Money >>> in school is very limited,so I am hoping to make one. Would it be >>> possible >>> for you to send me details on how to construct a simple tank,and whether >>> ordinary agar could be used instead of agarose as this would also cut >>> costs. >>> >> > Agarose for electrophoresis has been purified to contain few if any charged > groups, which would cause a phenomenon called electroendosmosis. This can > seriously degrade resolution. Exception: Counter-stream > immunoelectrophoresis and certain modes of capillary electrophoresis, where > electroendosmosis is actually required for separation. > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- letters.cunningprojects.com twitter.com/onetruecathal Kiva.org - Loans That Change Lives From gkapolas from yahoo.gr Mon Jun 29 04:47:06 2009 From: gkapolas from yahoo.gr (george kapolas) Date: Mon Jun 29 14:12:03 2009 Subject: agarose electrophoresis tank @ $10 Message-ID: <837035.99393.qm@web25008.mail.ukl.yahoo.com> Hi! Check this site also! http://www.scq.ubc.ca/the-macgyver-project-genomic-dna-extraction-and-gel-electrophoresis-experiments-using-everyday-materials/ Good luck! ___________________________________________________________ ?????????????? Yahoo!; ?????????? ?? ?????????? ???????? (spam); ?? Yahoo! Mail ???????? ??? ???????? ?????? ????????? ???? ??? ??????????? ????????? http://login.yahoo.com/config/mail?.intl=gr From taducute from gmail.com Mon Jun 29 12:45:51 2009 From: taducute from gmail.com (snehal k) Date: Mon Jun 29 14:14:44 2009 Subject: please help In-Reply-To: <1f50e3d20906290843n29beb600oe2131afb1d827a1f@mail.gmail.com> References: <1f50e3d20906290843n29beb600oe2131afb1d827a1f@mail.gmail.com> Message-ID: <1f50e3d20906291045p46f3486dlaaa790fa4a842e3b@mail.gmail.com> respected sir/madam i am student of microbiology ,mumbai university ,india. i am working on polyethylene glycol(PEG) degrading activity in my bacteria . i took ur work as reference.but sir i dont know how to sterilize PEG ,by autoclaving PEG or by filter sterilization of PEG. if you can tell me how to sterilize this i will be greatful to u. plz do the needful and oblidge. please help me out.. Regards, Snehal k From R.Jayakumar from roswellpark.org Mon Jun 29 15:35:03 2009 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Mon Jun 29 15:52:51 2009 Subject: please help In-Reply-To: <1f50e3d20906291045p46f3486dlaaa790fa4a842e3b@mail.gmail.com> References: <1f50e3d20906290843n29beb600oe2131afb1d827a1f@mail.gmail.com> <1f50e3d20906291045p46f3486dlaaa790fa4a842e3b@mail.gmail.com> Message-ID: I autoclave them routinely for my bacterial transformation work. But I am not sure which work you are referring to. But fillter sterilization is alsoa possibility. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of snehal k Sent: Monday, June 29, 2009 1:46 PM To: methods@iubio.bio.indiana.edu Subject: please help respected sir/madam i am student of microbiology ,mumbai university ,india. i am working on polyethylene glycol(PEG) degrading activity in my bacteria . i took ur work as reference.but sir i dont know how to sterilize PEG ,by autoclaving PEG or by filter sterilization of PEG. if you can tell me how to sterilize this i will be greatful to u. plz do the needful and oblidge. please help me out.. Regards, Snehal k _______________________________________________ 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 hroychow from nmsu.edu Mon Jun 29 16:54:28 2009 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Mon Jun 29 17:26:43 2009 Subject: please help In-Reply-To: <1f50e3d20906291045p46f3486dlaaa790fa4a842e3b@mail.gmail.com> References: <1f50e3d20906290843n29beb600oe2131afb1d827a1f@mail.gmail.com> <1f50e3d20906291045p46f3486dlaaa790fa4a842e3b@mail.gmail.com> Message-ID: <1390.128.123.174.0.1246312468.squirrel@webmail.nmsu.edu> PEG can be autoclaved. > respected sir/madam > i am student of microbiology ,mumbai university ,india. > i am working on polyethylene glycol(PEG) degrading activity in my bacteria > . > i took ur work as reference.but sir i dont know how to sterilize PEG ,by > autoclaving PEG or by filter sterilization of PEG. > > if you can tell me how to sterilize this i will be greatful to u. > > plz do the needful and oblidge. > > please help me out.. > > > Regards, > Snehal k > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Hiranya S. Roychowdhury, Ph.D. Asst. Professor, Health & Public Services Dona Ana Community College New Mexico State University Las Cruces, NM 88003 From hroychow from nmsu.edu Mon Jun 29 16:55:28 2009 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Mon Jun 29 17:26:59 2009 Subject: agarose electrophoresis tank @ $10 In-Reply-To: <468b1a400906290156u14a114a2r9d3b0c982d5a8c75@mail.gmail.com> References: <000501c9f648$1af48340$5d0d860a@BIDDULPH.INTERNAL> <468b1a400906290156u14a114a2r9d3b0c982d5a8c75@mail.gmail.com> Message-ID: <1403.128.123.174.0.1246312528.squirrel@webmail.nmsu.edu> We did that some 15 years back! > You might enjoy this article in the Science Creative Quarterly, which > explains how to use available-at-supermarket ingredients to perform DNA > extractions, gel electrophoresis and staining. > http://is.gd/1hPJs > The short answer is: you can use agar, though the band will be a little > blurry, and you can use a blue food colouring to stain the DNA, the > specific > name is in the article. Your agar might need to be good and pure, but if > it's just to demonstrate the principal of electrophoresis it's fine. > > 2009/6/26 Dr Engelbert Buxbaum > >> Am 26.06.2009, 13:21 Uhr, schrieb aria johnson : >> >> Hi Check this website out for simple tank >>> http://www.science-projects.com/GE/HorizChamber.htm >>> not sure whether agar will give you the resolution you need for the >>> exercise. >>> Will you use EtBr and UV for visualization? Im not sure what age is 6th >>> form >>> but you may want to monitor the EtBr step or find an alternate way >>> to visualize your pcr product. >>> take care >>> aj >>> >>> On Fri, Jun 26, 2009 at 6:23 AM, mccraight < >>> mccraight@biddulph.staffs.sch.uk >>> >>>> wrote: >>>> >>> >>> Hi, I am a Lab technician in a school,and we need an electophoresis >>> tank >>>> to >>>> enable our 6th for students to follow a new forensic science course. >>>> Money >>>> in school is very limited,so I am hoping to make one. Would it be >>>> possible >>>> for you to send me details on how to construct a simple tank,and >>>> whether >>>> ordinary agar could be used instead of agarose as this would also cut >>>> costs. >>>> >>> >> Agarose for electrophoresis has been purified to contain few if any >> charged >> groups, which would cause a phenomenon called electroendosmosis. This >> can >> seriously degrade resolution. Exception: Counter-stream >> immunoelectrophoresis and certain modes of capillary electrophoresis, >> where >> electroendosmosis is actually required for separation. >> >> _______________________________________________ >> Methods mailing list >> Methods@net.bio.net >> http://www.bio.net/biomail/listinfo/methods >> > > > > -- > letters.cunningprojects.com > twitter.com/onetruecathal > Kiva.org - Loans That Change Lives > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Hiranya S. Roychowdhury, Ph.D. Asst. Professor, Health & Public Services Dona Ana Community College New Mexico State University Las Cruces, NM 88003