From novalidaddress from nurfuerspam.de Mon Aug 4 05:25:59 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Mon Aug 4 09:13:30 2008 Subject: How to make polymerase stop in sequence? Message-ID: <05640717-17d1-41f8-bf28-657fe28c55f3@y38g2000hsy.googlegroups.com> Dear Experts, is there a way how one can make the DNA polymerase (ordinary Taq should do, no proofreading activity is desired) stop within a sequence introduced by a synthetic primer? I thought of something like several sugar building blocks without any base attached or a modified base which is so bulky that the pol must stop and slip off. The modified position does not need to align to the parent DNA strand as it may be placed in a loop. It just should be able to be synthesized with standard phosphoramidite technology. Many thanks for sharing your thoughts, Wo From bmacgreg from unc.edu Mon Aug 4 12:40:09 2008 From: bmacgreg from unc.edu (Barbara MacGregor) Date: Mon Aug 4 19:30:41 2008 Subject: Stopping Taq In-Reply-To: <200808041706.m74H64O25785@net.bio.net> References: <200808041706.m74H64O25785@net.bio.net> Message-ID: Hi, I'm not sure if this is exactly what you are looking for, it requires some synthesis, but maybe it will give you an idea: Chemistry Letters Vol. 37 (2008) , No. 6 p.584 Site-selective Termination of DNA Replication by Using a Caged Template Keita Tanaka, Akinori Kuzuya and Makoto Komiyama Barbara MacGregor > > > is there a way how one can make the DNA polymerase (ordinary Taq > should do, no proofreading activity is desired) stop within a sequence > introduced by a synthetic primer? I thought of something like several > sugar building blocks without any base attached or a modified base > which is so bulky that the pol must stop and slip off. > The modified position does not need to align to the parent DNA strand > as it may be placed in a loop. It just should be able to be > synthesized with standard phosphoramidite technology. > > Many thanks for sharing your thoughts, > > Wo > From n.zenkin from newcastle.ac.uk Mon Aug 4 12:02:09 2008 From: n.zenkin from newcastle.ac.uk (Nikolay Zenkin) Date: Mon Aug 4 19:30:48 2008 Subject: Imidazole and Amicon concentrators Message-ID: I used to concentrate by just ammonium sulphate precipitation with subsequent dialysis. Nikolay From baorui_ren from hotmail.com Mon Aug 4 21:28:20 2008 From: baorui_ren from hotmail.com (=?gb2312?B?sPzI8Q==?=) Date: Tue Aug 5 11:14:19 2008 Subject: HELP! protein-protein cross-linking Message-ID: Hello, AllWe have a problem in the chemical cross-linking experiment for protein-protein interaction. We have solved a protein complex structure, and we want to confirm their interaction by cross-linking. But i am a new comer in this field, the initial test was failed. Protein A is 35kd, Protein B is 14kd, but after cross-linking the band representing 49kd is very very weak in our SDS-PAGE comparing with the inact Protein A and Protein B in thesame lane. The condition we used is : add DSS to protein sample containing protein A and protein B and incubated for 1 hour under 4 degree, and then terminated it with 1M tris for 30 min. The samples were loaded on SDS-PAGE. We have tried different concentrations of DSS (0.1mM-10mM) and protein sample (20uM-1mM). PS: from the complex structure , we have found several Arg residues in both protein were located in the interface, so we use the DSS for initial test.Protein A trends to form dimer, but we could not even got the dimer bands after cross-linking. I would like to hear any suggestion from you.Thanks! From jgardn from lsuhsc.edu Tue Aug 5 13:39:05 2008 From: jgardn from lsuhsc.edu (Gardner, Jason D.) Date: Tue Aug 5 19:25:23 2008 Subject: Cytofluor II (was: "Fluoroscan II software") Message-ID: <9DE950516C27AB4FBD3DCECDC802AE895723F2@EXCHBE2.master.lsuhsc.edu> I was just hired at LSU and found a Cytofluor II fluorescent platereader in the Department. The software is nowhere to be found and I was wondering if someone could help me locate an install disk or manual. I think the computer left town after Hurricane Katrina a few years back. The Cytofluor appears to be in excellent condition. Thanks, Jason Gardner Department of Physiology Louisiana State University Health Sciences Center New Orleans From itisam.sarangi from gmail.com Wed Aug 6 04:34:29 2008 From: itisam.sarangi from gmail.com (Itisam Sarangi) Date: Wed Aug 6 10:34:53 2008 Subject: Info abt Antibody against Nuclear and cytoplasmic fraction Message-ID: <25de2fb70808060234u3e1ac764oa428a6732e2ae5a3@mail.gmail.com> hi all, I recently did a nuclear protein isolation from cultured cells. After western blot I can see my protein in nuclear and cytoplasmic fractions in equal concentration. So just to check the if there is any contamination of cytoplasmic protein in my nuclear fraction, I am interested to check them aganist an antibody. This antibody should be against a specific nuclear protein and should be present in every cell. I would appreciate any help in this case. If someone can suggest any antibody that can detect only a cytoplasmic protein also. If any one has a good established protocol for nuclear protein isolation please send me the link. thanking you for ur help -- Itisam Sarangi From matthias.leiser from agrobiogen.de Wed Aug 6 00:04:58 2008 From: matthias.leiser from agrobiogen.de (Matthias Leiser) Date: Wed Aug 6 10:35:38 2008 Subject: AW: Methods Digest, Vol 39, Issue 2 In-Reply-To: <200808051706.m75H5xO26229@net.bio.net> References: <200808051706.m75H5xO26229@net.bio.net> Message-ID: <3932D15FE7BC45DA85EF20DDA795EB5C@Labtop> Hi Wo, Look to the link below, maybe this gives you an idea how to solve your question. Best regards Matthias http://www.invitek.eu/content/forschung__technologie/technologien/molekulare _diagnostik/index_ger.html > is there a way how one can make the DNA polymerase (ordinary Taq > should do, no proofreading activity is desired) stop within a sequence > introduced by a synthetic primer? I thought of something like several > sugar building blocks without any base attached or a modified base > which is so bulky that the pol must stop and slip off. > The modified position does not need to align to the parent DNA strand > as it may be placed in a loop. It just should be able to be > synthesized with standard phosphoramidite technology. > > Many thanks for sharing your thoughts, > > Wo > ------------------------------ Message: 2 Date: Mon, 04 Aug 2008 19:55:48 GMT From: dk@no.email.thankstospam.net (DK) Subject: Re: How to make polymerase stop in sequence? To: methods@net.bio.net Message-ID: In article <05640717-17d1-41f8-bf28-657fe28c55f3@y38g2000hsy.googlegroups.com>, WS wrote: >Dear Experts, > >is there a way how one can make the DNA polymerase (ordinary Taq should >do, no proofreading activity is desired) stop within a sequence >introduced by a synthetic primer? I thought of something like several >sugar building blocks without any base attached or a modified base >which is so bulky that the pol must stop and slip off. >The modified position does not need to align to the parent DNA strand >as it may be placed in a loop. It just should be able to be synthesized >with standard phosphoramidite technology. Polymerase normally pops off when it encounters already present strand. So, how about throwing in another primer that anneals specifically to the sequence where you want the poly to stop? Having 3' phosphoramidite will prevent synthesis from this "killer" primer. Or make the 3' base dideoxy. Same thing. To prevent strand displacement, you can do extension with PfuUltra at 65C (or even below). Because I know no chemistry, that's what I'd do :-) DK ------------------------------ Message: 3 Date: Mon, 4 Aug 2008 18:02:09 +0100 From: Nikolay Zenkin Subject: Imidazole and Amicon concentrators To: "'methods@iubio.bio.indiana.edu'" Message-ID: Content-Type: text/plain; charset="us-ascii" I used to concentrate by just ammonium sulphate precipitation with subsequent dialysis. Nikolay ------------------------------ Message: 4 Date: Tue, 5 Aug 2008 02:28:20 +0000 From: =?gb2312?B?sPzI8Q==?= Subject: HELP! protein-protein cross-linking To: Message-ID: Hello, AllWe have a problem in the chemical cross-linking experiment for protein-protein interaction. We have solved a protein complex structure, and we want to confirm their interaction by cross-linking. But i am a new comer in this field, the initial test was failed. Protein A is 35kd, Protein B is 14kd, but after cross-linking the band representing 49kd is very very weak in our SDS-PAGE comparing with the inact Protein A and Protein B in thesame lane. The condition we used is : add DSS to protein sample containing protein A and protein B and incubated for 1 hour under 4 degree, and then terminated it with 1M tris for 30 min. The samples were loaded on SDS-PAGE. We have tried different concentrations of DSS (0.1mM-10mM) and protein sample (20uM-1mM). PS: from the complex structure , we have found several Arg residues in both protein were located in the interface, so we use the DSS for initial test.Protein A trends to form dimer, but we could not even got the dimer bands af! ter cross-linking. I would like to hear any suggestion from you.Thanks! ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 39, Issue 2 ************************************** From engelbert_buxbaum from hotmail.com Thu Aug 7 14:36:20 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Thu Aug 7 15:48:04 2008 Subject: HELP! protein-protein cross-linking References: Message-ID: Am 04.08.2008, 22:28 Uhr, schrieb ?? : > Hello, AllWe have a problem in the chemical cross-linking experiment for > protein-protein interaction. We have solved a protein complex structure, > and we want to confirm their interaction by cross-linking. Rather than using a kitchen knife (crosslinking) I'd prefer a laser skalpel for such applications (analytical ultracentrifugation, SPR, laser light scattering...) From Nikola.Wenta from nottingham.ac.uk Thu Aug 7 17:32:41 2008 From: Nikola.Wenta from nottingham.ac.uk (Nikola Wenta) Date: Fri Aug 8 11:14:24 2008 Subject: HELP! protein-protein cross-linking In-Reply-To: <200808051706.m75H5xO26229@net.bio.net> References: <200808051706.m75H5xO26229@net.bio.net> Message-ID: Hi! Maybe you can try crosslinking with glutaraldehyde. Do a time course over 1 hour with 400 ul Protein solution by adding 8 ul 1%(v/v) glutaraldehyde solution at room temperature. For each time point, take out 80 ul sample, add 8 ul 1 M Tris-HCl (pH 6.8) to terminate, add 24 ul 6x SDS sample buffer and load on a gel. Depending on the initial protein concentration, you may consider silver staining; gradient gel; are you sure that your protein should dimerize?? How can you know? What's the Kd value? That should be your initial protein concentration. Cheers, Niko Hello, AllWe have a problem in the chemical cross-linking experiment for protein-protein interaction. We have solved a protein complex structure, and we want to confirm their interaction by cross-linking. But i am a new comer in this field, the initial test was failed. Protein A is 35kd, Protein B is 14kd, but after cross-linking the band representing 49kd is very very weak in our SDS-PAGE comparing with the inact Protein A and Protein B in thesame lane. The condition we used is : add DSS to protein sample containing protein A and protein B and incubated for 1 hour under 4 degree, and then terminated it with 1M tris for 30 min. The samples were loaded on SDS-PAGE. We have tried different concentrations of DSS (0.1mM-10mM) and protein sample (20uM-1mM). PS: from the complex structure , we have found several Arg residues in both protein were located in the interface, so we use the DSS for initial test.Protein A trends to form dimer, but we could not even got the dimer bands af! ter cross-linking. I would like to hear any suggestion from you.Thanks! ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 39, Issue 2 ************************************** 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 Nikola.Wenta from nottingham.ac.uk Fri Aug 8 12:10:49 2008 From: Nikola.Wenta from nottingham.ac.uk (Nikola Wenta) Date: Fri Aug 8 14:37:09 2008 Subject: HELP! protein-protein cross-linking (Dr Engelbert Buxbaum) References: <200808081704.m78H4bU21819@net.bio.net> Message-ID: Hi again! I think that Dr. Buxbaum is absolutely right. Analytical Ultracentrifugation is definitely the method of choice: you can prove assoziation, get Kds, stoichiometries, even shapes ;-) You wrote that you have solved a structure; X-ray, right? Problem: solid crystal; what you try now is interactions in solution... You should also think about Static Light Scattering; often, SLS and AUC are used in parallel because they show similar things by different methods. Cheers, Niko This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation. From novalidaddress from nurfuerspam.de Sat Aug 9 05:40:42 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Sat Aug 9 13:07:08 2008 Subject: BLAST search of short ambiguous sequences? Message-ID: <04522ce8-e4bd-48ea-bf69-9178f6fd13ea@z72g2000hsb.googlegroups.com> Dear experts, I would like to search a genomic database for the occurrence of short motifs, like eg {Y,S,T} X {K,R}XX {Y,S,T} X means any amino acid, lists in {} are 2 or 3 amino acids at a specific position. Instead of generating all possible combinations and blasting them separately, is there a smarter way to do it? I couldn't find any hints in the BLAST documentation at NCBI Many thanks, Wo From e_barouki from hotmail.com Mon Aug 11 07:30:40 2008 From: e_barouki from hotmail.com (Emad albarouki) Date: Mon Aug 11 11:15:28 2008 Subject: Methods Digest, Vol 39, Issue 5 In-Reply-To: <200808091703.m79H3lU19240@net.bio.net> References: <200808091703.m79H3lU19240@net.bio.net> Message-ID: Dear Experts, I was wondering if the OliC promoter from Aspergillus nidulans, which is often used for antibiotic resistant cassettes in fungal gene disrupting vectors. Can this promoter be used for expression in E coli! Does anybody have an idea about that? Thanks in Advance EB _________________________________________________________________ Get more from your digital life. Find out how. http://www.windowslive.com/default.html?ocid=TXT_TAGLM_WL_Home2_082008 From rashmi21481 from gmail.com Mon Aug 11 10:30:57 2008 From: rashmi21481 from gmail.com (rashmi) Date: Mon Aug 11 11:15:35 2008 Subject: high percentage agarose Message-ID: <42c986dd-0c96-4fa2-bbef-d79a4d751cb3@o40g2000prn.googlegroups.com> Hi there, I am a PhD student working on Pneumocystis biodiversity. I cam across your message posted on google groups, regarding high percentage agarose gels and TBE concentration. One of the objective of my thesis is to do typing by counting number of 10 base pair repeats in my PCR products. The number of repeats can vary from 2 to 6. The sizes are 125, 135, 145, 155 and 165. I tried to run a 4% agarose gel on a tray about 25 cm. But my gel was not properly formed. i will be highly delighted if you could suggest me something, like the % of agarose I should use, run time, gel lengh, voltage etc. Thanks in advance Rashmi Gupta PhD Student AIIMS New Delhi From novalidaddress from nurfuerspam.de Mon Aug 11 11:45:59 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Mon Aug 11 16:21:40 2008 Subject: high percentage agarose References: <42c986dd-0c96-4fa2-bbef-d79a4d751cb3@o40g2000prn.googlegroups.com> Message-ID: Dear Rashmi, If you (or your PI) insist(s) on agarose, you might need some special grade suitable for high percentage gels. Do you have the possibility to run Acrylamide gels instead? Then you might use silver staining to visualize the DNA. Best regards, Wolfgang From ohamarsheh from gmail.com Mon Aug 11 14:38:45 2008 From: ohamarsheh from gmail.com (Dr Omar Hamarsheh) Date: Mon Aug 11 16:22:01 2008 Subject: high percentage agarose In-Reply-To: <42c986dd-0c96-4fa2-bbef-d79a4d751cb3@o40g2000prn.googlegroups.com> References: <42c986dd-0c96-4fa2-bbef-d79a4d751cb3@o40g2000prn.googlegroups.com> Message-ID: <000a01c8fbe9$f41b22f0$dc5168d0$@com> Dear Rashmi, I suggest on you to use MetaPhore agarose, 4.5 % will be very good for small fragments. Be aware that you need to cook it carefully. Fist use cold buffer (stored on 4C), add the MetaPhore garose powder little by little while mixing using magnetic stirrer, let the mix for 15 minutes then cook it in the microwave starting at low heat then increase the temperature gradually (it needs 20- 30 min ). For running the gel, 150 watts for 4-5 hours, do not put stain (Ethedium bromide) with the gel, just incubate the gel after running for 15-20 min in a bath of Eth. Brd diluted with distilled water All the best, Omar -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of rashmi Sent: Monday, August 11, 2008 6:31 PM To: methods@magpie.bio.indiana.edu Cc: rashmi21481@gmail.com Subject: high percentage agarose Hi there, I am a PhD student working on Pneumocystis biodiversity. I cam across your message posted on google groups, regarding high percentage agarose gels and TBE concentration. One of the objective of my thesis is to do typing by counting number of 10 base pair repeats in my PCR products. The number of repeats can vary from 2 to 6. The sizes are 125, 135, 145, 155 and 165. I tried to run a 4% agarose gel on a tray about 25 cm. But my gel was not properly formed. i will be highly delighted if you could suggest me something, like the % of agarose I should use, run time, gel lengh, voltage etc. Thanks in advance Rashmi Gupta PhD Student AIIMS New Delhi _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From peter.ianakiev from gmail.com Mon Aug 11 15:56:38 2008 From: peter.ianakiev from gmail.com (peter) Date: Mon Aug 11 16:22:07 2008 Subject: high percentage agarose References: <42c986dd-0c96-4fa2-bbef-d79a4d751cb3@o40g2000prn.googlegroups.com> Message-ID: On Aug 11, 4:21 pm, d...@no.email.thankstospam.net (DK) wrote: > In article <42c986dd-0c96-4fa2-bbef-d79a4d751...@o40g2000prn.googlegroups.com>, rashmi wrote: > > >Hi there, > >I am a PhD student working on Pneumocystis biodiversity. I cam across > >your message posted on google groups, regarding high percentage > >agarose gels and TBE concentration. One of the objective of my thesis > >is to do typing by counting number of 10 base pair repeats in my PCR > >products. The number of repeats can vary from 2 to 6. The sizes are > >125, 135, 145, 155 and 165. I tried to run a 4% agarose gel on a tray > >about 25 cm. But my gel was not properly formed. i will be highly > >delighted if you could suggest me something, like the % of agarose I > >should use, run time, gel lengh, voltage etc. > > This is definitely a job for acrylamide gel. Google a bit and you > shall find. > > DK I have done in the pas discrimination of 3 bp 126/129 allele on 6% agarose. The trick was to boil agarose for long time and to add some urea in it while is boiling. So if you need to make 100 ml 6% agarose start with 200 ml H2O +10ml TBE + 6g Agarose, when is boiling add 1-2 g Urea, keep boiling until volume is reduced to 100 ml and immediately pour without cooling. Don't worry about bubbles, they will come out. One more thing - run the gel as fast as you can (high voltage /cm) . Good luck From hroychow from nmsu.edu Mon Aug 11 16:22:02 2008 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Mon Aug 11 19:30:38 2008 Subject: high percentage agarose In-Reply-To: <42c986dd-0c96-4fa2-bbef-d79a4d751cb3@o40g2000prn.googlegroups.com> References: <42c986dd-0c96-4fa2-bbef-d79a4d751cb3@o40g2000prn.googlegroups.com> Message-ID: <2130.71.210.217.92.1218489722.squirrel@webmail.nmsu.edu> The opening line always fascinates me! The email was directed to the methods listserve, and to be sure, there would be someone in this group wo had posted on some unspecified "google groups"! Anyway, I suggest a mini acrylamide sequencing set-up. There are many protocols for that in places as you have mentioned. > Hi there, > I am a PhD student working on Pneumocystis biodiversity. I cam across > your message posted on google groups, regarding high percentage > agarose gels and TBE concentration. One of the objective of my thesis > is to do typing by counting number of 10 base pair repeats in my PCR > products. The number of repeats can vary from 2 to 6. The sizes are > 125, 135, 145, 155 and 165. I tried to run a 4% agarose gel on a tray > about 25 cm. But my gel was not properly formed. i will be highly > delighted if you could suggest me something, like the % of agarose I > should use, run time, gel lengh, voltage etc. > Thanks in advance > Rashmi Gupta > PhD Student > AIIMS > New Delhi > _______________________________________________ > 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 aawara from pontiff-playground.org Mon Aug 11 20:25:56 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Mon Aug 11 23:31:55 2008 Subject: BLAST search of short ambiguous sequences? References: <04522ce8-e4bd-48ea-bf69-9178f6fd13ea@z72g2000hsb.googlegroups.com> Message-ID: In <04522ce8-e4bd-48ea-bf69-9178f6fd13ea@z72g2000hsb.googlegroups.com>, WS wrote: > Dear experts, > > I would like to search a genomic database for the occurrence of short > motifs, like eg {Y,S,T} X {K,R}XX {Y,S,T} > X means any amino acid, lists in {} are 2 or 3 amino acids at a > specific position. > > Instead of generating all possible combinations and blasting them > separately, is there a smarter way to do it? I couldn't find any hints > in the BLAST documentation at NCBI If you have a few sequences that match this consensus, then you can do a PHI BLAST search. I just did this a little while ago, and it worked well. My pattern was about 8 a.a. long. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From agbiok4 from gmail.com Mon Aug 11 22:50:22 2008 From: agbiok4 from gmail.com (kamalaker nasani) Date: Mon Aug 11 23:32:03 2008 Subject: Fwd: protein purification In-Reply-To: References: Message-ID: ---------- Forwarded message ---------- From: kamalaker nasani Date: Aug 12, 2008 9:19 AM Subject: protein purification To: methods@net.bio.net Hi I need to concentrate 66KDa size protein in my sample.(from plant coded by transgene) Can any one give the suggestions regarding this What concentraion I have to go for Ammonium sulphate saturation and any other method is there to purify? -- URS Kamalaker Nasani "The illiterate of the 21st Century will not be those who cannot read or write. The illiterate will be those who cannot learn, unlearn and relearn" -George Bernard Shaw -- URS Kamalaker Nasani "The illiterate of the 21st Century will not be those who cannot read or write. The illiterate will be those who cannot learn, unlearn and relearn" -George Bernard Shaw From M.Dunowska from massey.ac.nz Mon Aug 11 19:57:17 2008 From: M.Dunowska from massey.ac.nz (Dunowska, Magda) Date: Mon Aug 11 23:32:12 2008 Subject: high percentage agarose In-Reply-To: <2130.71.210.217.92.1218489722.squirrel@webmail.nmsu.edu> References: <42c986dd-0c96-4fa2-bbef-d79a4d751cb3@o40g2000prn.googlegroups.com> <2130.71.210.217.92.1218489722.squirrel@webmail.nmsu.edu> Message-ID: <92FDFD8B26EB6542B1E1BF017BB998D14F4E5EA3C0@TUR-EXCHMBX.massey.ac.nz> A long time ago I was looking for small differences between fragments and used pre-cast polyacrylamide gels (from Bio-Rad I think), which I stained with a gel-star nucleic acid stain (currently sold by Lonza). Worked really well - Magda Magda Dunowska, LW (vet), PhD Senior Lecturer in Veterinary Infectious Diseases (Virology) Institute of Veterinary, Animal and Biomedical Sciences Te Kura Mātauranga Kararehe Massey University Palmerston North New Zealand Phone : (06) 356-9099 ext 7571 Website : http://ivabs.massey.ac.nz -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Dr. Hiranya S. Roychowdhury Sent: Tuesday, 12 August 2008 9:22 a.m. To: rashmi Cc: methods@magpie.bio.indiana.edu; rashmi21481@gmail.com Subject: Re: high percentage agarose The opening line always fascinates me! The email was directed to the methods listserve, and to be sure, there would be someone in this group wo had posted on some unspecified "google groups"! Anyway, I suggest a mini acrylamide sequencing set-up. There are many protocols for that in places as you have mentioned. > Hi there, > I am a PhD student working on Pneumocystis biodiversity. I cam across > your message posted on google groups, regarding high percentage > agarose gels and TBE concentration. One of the objective of my thesis > is to do typing by counting number of 10 base pair repeats in my PCR > products. The number of repeats can vary from 2 to 6. The sizes are > 125, 135, 145, 155 and 165. I tried to run a 4% agarose gel on a tray > about 25 cm. But my gel was not properly formed. i will be highly > delighted if you could suggest me something, like the % of agarose I > should use, run time, gel lengh, voltage etc. > Thanks in advance > Rashmi Gupta > PhD Student > AIIMS > New Delhi > _______________________________________________ > 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 _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From agbiok4 from gmail.com Tue Aug 12 05:11:45 2008 From: agbiok4 from gmail.com (kamalaker nasani) Date: Tue Aug 12 12:17:57 2008 Subject: protein work Message-ID: Hi I need to concentrate 66KDa size protein in my sample.(from plant coded by transgene) Can any one give the suggestions regarding this What concentraion I have to go for Ammonium sulphate saturation and any other method is there to purify? -- URS Kamalaker Nasani -- URS Kamalaker Nasani "The illiterate of the 21st Century will not be those who cannot read or write. The illiterate will be those who cannot learn, unlearn and relearn" -George Bernard Shaw From davidminde from gmail.com Tue Aug 12 07:31:30 2008 From: davidminde from gmail.com (David-Paul Minde) Date: Tue Aug 12 12:18:17 2008 Subject: mutagenesis Message-ID: <123243900808120531j7a3abfd4rb406283435f880f5@mail.gmail.com> ... (hope you have no longer any problems with mutagenesis as in a post some 6 years back but ..) I strongly prefer Phusion (=highest fidelity commercially available polymerase) PCR for mutagenesis. Piece of cake easy for up to ~20 kb plasmids :) ... best, David -- 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 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 dprieto from fcien.edu.uy Wed Aug 13 09:47:19 2008 From: dprieto from fcien.edu.uy (Daniel Prieto) Date: Wed Aug 13 12:52:57 2008 Subject: high percentage agarose Message-ID: <48A2F3F7.7000809@fcien.edu.uy> Hi, I agree with DK. I would suggest you to run a 6% acrylamide gel and silver staining, since your fragments are very close and small so this would give you the best resolution and it's pretty straightforward. If you don't have any recipes for that, just ask ;). Good luck! Daniel From Sharon.Waldrop from utsouthwestern.edu Wed Aug 13 18:16:38 2008 From: Sharon.Waldrop from utsouthwestern.edu (Sharon Waldrop) Date: Thu Aug 14 09:37:24 2008 Subject: Western background staining with Primary antibody (Rabbit) Message-ID: <48A325060200003E000368DB@swnw126.swmed.org> Hello Group. When I was working with Drosophila embryos, we would preincubate the primary antibody (overnight with rotation at 4 degrees) with embryos that did not have the antigen of interest thus taking care of background (worked great). Now I am working with cell lines. With my primary antibody to cell surface receptor, I find that if I fix cells that do not have receptor (100 million) with 4%pfa and then wash the cells with PBS a couple of times. I then add 100ul of primary antibody, put on rotator overnight at 4 degrees and bye-bye background on lysed/treated plasma membranes. Westerns look great. What can I do about an antibody against an intracellular component in the cytoplasm (rabbit, of course)? I am having to use the antibody/5% dry milk/TBST 4 to 5 western run times on Nitrocellulose to get rid of the background before I get clear bands. Please do not suggest PDVF membrane (10X worse). I have also tried different concentrations of antibody (no difference). It is not the secondary, already looked at that as the cause of problem (use it with the extracellular - works fine). Pulling my hair out as well as wasting time and reagents. Any suggestions? Thanks. From blackhole from abuse.plus.com Thu Aug 14 03:27:15 2008 From: blackhole from abuse.plus.com (Duncan Clark) Date: Thu Aug 14 09:37:31 2008 Subject: mutagenesis References: Message-ID: Historians believe that in newspost on Tue, 12 Aug 2008, DK penned the following literary masterpiece: >Basically, Stratagene stole Finnzyme's idea, somehow >managed to circumvent IP (patent) protection and ended >up making a better product. MJ Bioworks (prior to MJ's downfall over the thermal cycler infringement with ABI) patented Sac 7/Sso7 (7kd non-specific ds DNA binding protein from Sulfolobus) as a fusion with a DNA polymerase. They then specified in another patent that one had to have various aa's at positions a ... z in the Sso sequence in order to bind DNA. Stratagene basically altered every other amino acid else bar those and after lots of screening identified a modified Sso7 that worked as well as the wt Sso7 in a fusion. All fun and games :-) Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From n.mvenge from imperial.ac.uk Thu Aug 14 07:40:20 2008 From: n.mvenge from imperial.ac.uk (Mvenge, Nyasha) Date: Thu Aug 14 09:37:45 2008 Subject: Hygromycin as a marker for Non tuberculous mycobacteria Message-ID: <1CA1A859632B1F4989CB23F90D0A7829042C33BA@icex4.ic.ac.uk> I am writting to ask about what concentrations you have found to be effective when selecting for transformants of Non tuberculous mycobacteria such as M.avium,abscessus and chelonae. Currently we have been using 50 and 100 ucl/ml but we keep getting colonies growing effectively rendering selection a complete waste of time.Apparently selection should work at 50ucl/ml.Do you have any suggestions for any other antibiotic apart from kanamycin? Thank you Grace. From sschneider from cylex.net Thu Aug 14 08:28:54 2008 From: sschneider from cylex.net (Schneider, Steve) Date: Thu Aug 14 09:37:50 2008 Subject: Low Biotin BSA\ Message-ID: <4EAD17449E7D4A4DBFB70C38037E5DEB5CAB20@mail-dcfs.hqcorp.cylex.net> Wolfgang, I stumbled upon your email today and have been researching the same problem of finding a BSA with low biotin. SIGMA has such a product. It is Cat No. A4503. Steven Schneider Senior Scientist Cylex, Inc. 8980-I Old Annapolis Rd. Columbia, MD 21045 Phone: (410) 964-0236 Fax: (410) 964-0367 Email: sschneider@cylex.net Internet: www.cylex.net From andrew.wright from tufts.edu Thu Aug 14 15:20:46 2008 From: andrew.wright from tufts.edu (Andrew Wright) Date: Thu Aug 14 22:14:19 2008 Subject: Looking for plasmid vector with no pUC sequences Message-ID: Dear David Steffen, I noticed on the internet that you may have the DNA sequence of plasmid pGB2. I am using this plasmid and would be very grateful if you could send me the sequence, should you have it. Sincerely, Andrew wright Andrew Wright, Ph.D. Professor Department of Molecular Biology Tufts Medical School 136 Harrison Ave. Boston, MA 02111 Phone: 617-636-6760 FAX: 617-636-0337 From rashmi1208 from googlemail.com Mon Aug 18 07:32:40 2008 From: rashmi1208 from googlemail.com (Rashmi Srivastava) Date: Mon Aug 18 16:04:38 2008 Subject: charge on protein Message-ID: <7a51f5da0808180532l41fa0046y8fbba3ef1a49d8a0@mail.gmail.com> Hello Experts, What simple methods can I use to determine the overall charge on a membrane protein?? Rashmi From novalidaddress from nurfuerspam.de Mon Aug 18 17:52:55 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Tue Aug 19 13:52:50 2008 Subject: charge on protein References: Message-ID: Dear Rashmi, you could use the "pI Tool" from Expasy as long as you know your sequence. http://www.expasy.org/ Of course, when your protein is modified the results need corrections. A better way might be a 2D Gel (or at least 1D-isoelectric focusing) where you may determine the *real* state of your buddy. Best regards, Wo On 18 Aug., 14:32, "Rashmi Srivastava" wrote: > Hello Experts, > > What simple methods can I use to determine the overall charge on a membrane > protein?? > > Rashmi From ASchmidt from ice.mpg.de Tue Aug 19 08:41:19 2008 From: ASchmidt from ice.mpg.de (Axel Schmidt) Date: Tue Aug 19 13:53:23 2008 Subject: Qaigen + kits etc Message-ID: <48AACD7F.2030808@ice.mpg.de> -- ----------- Dr. Axel Schmidt Max-Planck-Institute for Chemical Ecology Hans-Knoell-Str.8 Beutenberg-Campus D-07745 Jena Tel: 03641 / 571331 Fax: 03641 / 571302 From engelbert_buxbaum from hotmail.com Tue Aug 19 08:35:33 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Aug 19 13:53:28 2008 Subject: Fwd: protein purification References: Message-ID: Am 11.08.2008, 23:50 Uhr, schrieb kamalaker nasani : > I need to concentrate 66KDa size protein in my sample.(from plant coded > by > transgene) > > Can any one give the suggestions regarding this > > What concentraion I have to go for Ammonium sulphate saturation and any > other method is there to purify? There is no good correlation between molecular mass and the AS concentration required for precipitation. You need to determine the maximal concentration at which your protein does not precipitate and the minimal conc for total precipitation in a series (say, every 10%). If you have an antibody against your protein, spot-blots of supernatants and precipitates are the way to go. But for simple concentration (rather than purification) however I'd probably use ultrafiltration, not AS precipitation. Amicon pressure cells (say, with a 30 kDa membrane) do that neetly and are available for volumes from a few ml to several l. From engelbert_buxbaum from hotmail.com Tue Aug 19 08:44:11 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Aug 19 13:53:34 2008 Subject: Western background staining with Primary antibody (Rabbit) References: <_MXok.13253$Bt6.3194@newsfe04.iad> Message-ID: Am 14.08.2008, 11:01 Uhr, schrieb DK : > The best (but more laborous) thing to do: > > Extract cytosolic proteins from the cells (hypotonic lysis/dounce), > spin in ultracentrifuge to remove all insolubles, concentrate to > at least 5 mg/ml protein, dialize *extensively* (to remove small > molecules) against secondary amine-free buffer, couple to > 1-3 ml of an immobilized support (1:1 mixture of Bio-Rad's AffiGel 10 > and 15 would work best; 5-10 mg/ml of activated gel), pack > into a small column, then run your antibodies over it (reload > flow through to be sure). Zero background guaranteed. However, you may get away with just incubating the cytosol with a membrane rather than doing a coupling reaction. That should be a lot quicker and cheaper. In this case however I would prefer PVDF over NC because of the stronger protein binding. From engelbert_buxbaum from hotmail.com Tue Aug 19 08:58:03 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Aug 19 13:53:42 2008 Subject: charge on protein References: Message-ID: Am 18.08.2008, 08:32 Uhr, schrieb Rashmi Srivastava : > What simple methods can I use to determine the overall charge on a > membrane protein?? There is none. For a review on the problem see D.J. Winzor: Determination of the net charge (valence) of a protein: a fundamental but elusive parameter, Anal.Biochem. 325 (2004) 1-20. Be particularly warry about all these naive computer programs that claim to calculate charge or pI from sequence. The pKa values tabulated for amino acid side chains are valid only in aqueous solution. Inside a protein the pKa may differ by 2-3 pH-units because of hydrophobic environment and the presence of other charged residues. This is of fundamental importance for the reaction mechanism of many enzymes. Example: Bacillus circulans xylanase Glu-172 pKa = 6.7 acts as proton donor for acid/base catalysis, Glu-78 pKa = 4.6 is ionised and stabilises the positively charged intermediate. From mlsulliv from wisc.edu Wed Aug 20 09:02:53 2008 From: mlsulliv from wisc.edu (Michael Sullivan) Date: Wed Aug 20 10:26:20 2008 Subject: Postdoc opportunity-Madison, WI Message-ID: My lab at the US Dairy Forage Research Center in Madison, WI has =20 recently been awarded a USDA-CSREES-NRI grant to support our research =20= on the biochemical pathways responsible for o-diphenol biosynthesis =20 in red clover. This grant includes support for a postdoc position to =20 start in early 2009. Qualified candidates will have hands-on =20 experience in general molecular biology, including PCR, cloning, =20 sequencing, making gene constructs; biochemistry, including =20 characterization of proteins and analyses of enzymatic activities; =20 and/or plant transformation including plant tissue culture and =20 maintenance of transgenic plants. Candidates should also be able to =20 work independently and have good oral and written communication =20 skills. NON-US CITIZEN MAY BE ELIGIBLE FOR THIS POSITION, provided =20 they already have documentation allowing them to work in the US (I =20 CANNOT help with obtaining work visas). Non-US citizens must also =20 meet certain other eligibility requirements. Interested individuals may contact me by e-mail =20 (michael.sullivan@ars.usda.gov). The formal application process is =20 expected to begin in Dec. 2008. The US Dairy Forage Research Center is part of the Agricultural =20 Research Service of the US Department of Agriculture and is located =20 on the University of Wisconsin campus in Madison, WI. For more =20 information on the US Dairy Forage Research Center visit our web =20 site: http://ars.usda.gov/mwa/madison/dfrc Title: Elucidating the Roles of Hydroxycinnamoyl Transferases and p-=20 Coumaroyl 3-Hydroxylases in o-Diphenol Biosynthesis in Red Clover PD: Sullivan, Michael L. Institution: US Dairy Forage =20 Research Center, ARS-USDA Phenylpropanoid o-diphenols accumulate in tissues of many plants =20 functioning as defensive molecules and antioxidants. Red clover =20 accumulates high levels of two o-diphenols, phasalic acid and =20 clovamide. In red clover, post-harvest oxidation of these o-diphenols =20= to o-quinones by an endogenous polyphenol oxidase (PPO) prevents =20 breakdown of forage protein during storage. Agronomically important =20 forages like alfalfa lack both PPO and o-diphenols. Consequently, =20 breakdown of their protein upon harvest and storage results in =20 economic losses ($100 million/yr) and release of excess nitrogen into =20= the environment. Understanding how red clover is able to synthesize =20 and accumulate o-diphenols will help in development of forages that =20 take advantage of this natural system of protein protection. Also, =20 because o-diphenols are powerful antioxidants, this research has =20 implications for human and animal nutrition. Preliminary evidence =20 suggests that specific hydroxycinnamoyl transferases (HCTs) and p-=20 coumaroyl 3=92 hydroxylases (C3Hs) play key roles in red clover o-=20 diphenol biosynthesis and accumulation. Specific objectives of this =20 proposal are 1) Identify and isolate red clover gene sequences =20 encoding HCTs; 2) Characterize the HCTs and a C3H with respect to =20 substrate specificity and reaction characteristics; and 3) Establish =20 the relevance of specific HCTs to biosynthesis and accumulation of =20 specific o-diphenols. These objectives will be accomplished using =20 several complementary approaches including biochemistry, genomics, =20 and reverse genetics. --- Michael L. Sullivan Plant Research Molecular Geneticist US Dairy Forage Research Center ARS-USDA 1925 Linden Drive West Madison, WI 53706 (608) 890-0046 (Phone) (608) 890-0076 (FAX) From ddilernia from fmed.uba.ar Wed Aug 20 11:01:10 2008 From: ddilernia from fmed.uba.ar (Dario Dilernia) Date: Thu Aug 21 11:59:14 2008 Subject: Query about Maximum PCR product size for pGEM-T Message-ID: <000b01c902dd$f687e960$e397bc20$@uba.ar> Dear Tony Schountz, My name is Dario and I am from Argentina. I am sending you this mail because I am looking for information regarding the maximum PCR fragment size that can be cloned into pGEM-T Easy vector and I found on the internet that you have asked the same question in a web forum although I didn?t find whether somebody answered you Did you find this information? It would be very helpful for me. I see that your question was posted in the year 2000 so may be you already have experience in this field. Did you have success cloning large fragments with pGEM-T? Best regards, Dario Lic. Dario A. Dilernia Centro Nacional de Referencia para el SIDA Departamento de Microbiolog?a, Facultad de Medicina - UBA Paraguay 2155 Piso 11? - C1121ABG Buenos Aires, ARGENTINA Tel: 54 11 4508-3671/3689 int: 132 Fax: 54 11 4508-3705 -- Internal Virus Database is out-of-date. Checked by AVG Free Edition. Version: 7.5.432 / Virus Database: 268.16.2/613 - Release Date: 01/01/2007 02:50 p.m. From joelschneider914 from gmail.com Thu Aug 21 13:30:21 2008 From: joelschneider914 from gmail.com (Joel Schneider) Date: Thu Aug 21 19:11:24 2008 Subject: Protein Extraction from Fat Message-ID: <001b01c903bb$f9974d60$ecc5e820$@com> Hi all, I am currently interested in analyzing the protein content of a fat sample I recently harvested. Is there a particular protein extraction method recommended for fat? Thank you! ~J From chempalo from savba.sk Fri Aug 22 06:36:51 2008 From: chempalo from savba.sk (PavolFarkas) Date: Fri Aug 22 10:09:27 2008 Subject: BSA organic solvent Message-ID: <48AEA4D3.8070806@savba.sk> *Dear Mr. Wolfgang Schechinger! Did you find some organic solvents for BSA or Organic/water solvents? Thank you for answer, Your sincerely, Pavol Farkas * From biotech2020 from gmail.com Fri Aug 22 09:50:17 2008 From: biotech2020 from gmail.com (sankar jagarlamudi) Date: Fri Aug 22 10:09:33 2008 Subject: Methods Digest, Vol 39, Issue 14 In-Reply-To: <200808211706.m7LH63U07012@net.bio.net> References: <200808211706.m7LH63U07012@net.bio.net> Message-ID: <9d27670b0808220750i5aac8ad9wf16d506582575703@mail.gmail.com> Respected Sir/ Madam, i register for this subscription and i dont know how to operat= e this for my correspondence. Please let me know how to operate this journal. Thanks On Thu, Aug 21, 2008 at 10:36 PM, wrot= e: > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. Query about Maximum PCR product size for pGEM-T (Dario Dilernia) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 20 Aug 2008 13:01:10 -0300 > From: "Dario Dilernia" > Subject: Query about Maximum PCR product size for pGEM-T > To: > Message-ID: <000b01c902dd$f687e960$e397bc20$@uba.ar> > Content-Type: text/plain; charset=3D"iso-8859-1" > > Dear Tony Schountz, > > > > My name is Dario and I am from Argentina. I am sending you this mail > because > I am looking for information regarding the maximum PCR fragment size that > can be cloned into pGEM-T Easy vector and I found on the internet that yo= u > have asked the same question in a web forum although I didn=B4t find whet= her > somebody answered you=85 Did you find this information? It would be very > helpful for me. I see that your question was posted in the year 2000 so m= ay > be you already have experience in this field. Did you have success clonin= g > large fragments with pGEM-T? > > > > Best regards, > > > > Dario > > > > > > > > > > Lic. Dario A. Dilernia > > > > Centro Nacional de Referencia para el SIDA > > Departamento de Microbiolog=EDa, Facultad de Medicina - UBA > > Paraguay 2155 Piso 11=BA - C1121ABG > > Buenos Aires, ARGENTINA > > Tel: 54 11 4508-3671/3689 int: 132 > > Fax: 54 11 4508-3705 > > > > > > > -- > Internal Virus Database is out-of-date. > Checked by AVG Free Edition. > Version: 7.5.432 / Virus Database: 268.16.2/613 - Release Date: 01/01/200= 7 > 02:50 p.m. > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 39, Issue 14 > *************************************** > From davidminde from gmail.com Thu Aug 21 15:11:00 2008 From: davidminde from gmail.com (David-Paul Minde) Date: Fri Aug 22 10:10:03 2008 Subject: (no subject) Message-ID: <123243900808211311r224a52b2ra86987fb34c0183d@mail.gmail.com> I did not succed in cloning a 8.5 kb amplicon (after gel-extraction of Phusion PCR). pCR-XL TOPO (of invitrogen), however, worked fine in first attempt. cheers, David -- 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 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 novalidaddress from nurfuerspam.de Fri Aug 22 10:48:46 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Fri Aug 22 17:35:22 2008 Subject: BSA organic solvent References: Message-ID: <03a9826c-2905-4825-95c5-e87e575aa47d@b1g2000hsg.googlegroups.com> Hello Pavol, you might try Formamide (dissolves more, but takes longer) or DMSO. Takes lots of time. Just had a look on the shelf where some flasks reside already for 3years ;-) and indeed, in Formamide, about 1gram/ 50ml had completely dissolved. The solution has a funny aspect as it is somehow a little gelly. I haven't tried to add water as I wanted to avoid aqueous conditions. Ionic liquids might work, too. Besr regards and good luck! Wo From shaakanin from yahoo.com Fri Aug 22 21:56:00 2008 From: shaakanin from yahoo.com (Jess) Date: Sat Aug 23 12:59:17 2008 Subject: Methods Digest, Vol 39, Issue 14 Large PCR p-GEM In-Reply-To: <200808211706.m7LH63U07012@net.bio.net> Message-ID: <279262.22943.qm@web52910.mail.re2.yahoo.com> The largest fragment I was ever able to clone was about 3 kB. I am not a cl= oning master, and maybe someone else managed to get a bigger one, at some p= oint. We did not use Taq for the PCR, we used KOD polymerase to generate th= e PCR product, then A-tailed using Taq before ligation.=20 Cheers! Jess =A0 Grad. Res. Asst. University of Texas- Austin Institute for Cel= lular and Molecular Biology Department of Medicinal Chemistry =A0=A0=A0= =A0=A0=A0=A0=A0=A0=A0 -Hook 'em Horns! =A0 --- On Thu, 8/21/08, methods-request@oat.bio.indiana.edu wrote: From: methods-request@oat.bio.indiana.edu Subject: Methods Digest, Vol 39, Issue 14 To: methods@magpie.bio.indiana.edu Date: Thursday, August 21, 2008, 12:06 PM Send Methods mailing list submissions to =09methods@net.bio.net To subscribe or unsubscribe via the World Wide Web, visit =09http://www.bio.net/biomail/listinfo/methods or, via email, send a message with subject or body 'help' to =09methods-request@net.bio.net You can reach the person managing the list at =09methods-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. Query about Maximum PCR product size for pGEM-T (Dario Dilernia) ---------------------------------------------------------------------- Message: 1 Date: Wed, 20 Aug 2008 13:01:10 -0300 From: "Dario Dilernia" Subject: Query about Maximum PCR product size for pGEM-T To: Message-ID: <000b01c902dd$f687e960$e397bc20$@uba.ar> Content-Type: text/plain;=09charset=3D"iso-8859-1" Dear Tony Schountz, =20 My name is Dario and I am from Argentina. I am sending you this mail becaus= e I am looking for information regarding the maximum PCR fragment size that can be cloned into pGEM-T Easy vector and I found on the internet that you have asked the same question in a web forum although I didn=B4t find whethe= r somebody answered you=85 Did you find this information? It would be very helpful for me. I see that your question was posted in the year 2000 so may be you already have experience in this field. Did you have success cloning large fragments with pGEM-T? =20 Best regards, =20 Dario =20 =20 =20 =20 Lic. Dario A. Dilernia =20 Centro Nacional de Referencia para el SIDA Departamento de Microbiolog=EDa, Facultad de Medicina - UBA Paraguay 2155 Piso 11=BA - C1121ABG=20 Buenos Aires, ARGENTINA Tel: 54 11 4508-3671/3689 int: 132 Fax: 54 11 4508-3705 =20 =20 --=20 Internal Virus Database is out-of-date. Checked by AVG Free Edition. Version: 7.5.432 / Virus Database: 268.16.2/613 - Release Date: 01/01/2007 02:50 p.m. =20 ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 39, Issue 14 *************************************** =0A=0A=0A From sayeedansarmaricar from gmail.com Sat Aug 23 04:46:20 2008 From: sayeedansarmaricar from gmail.com (sayeedansarmaricar@gmail.com) Date: Sat Aug 23 13:00:01 2008 Subject: KCl in RT-PCR Message-ID: hi, can anyone tell me why do we use KCl in the reaction buffer for cDNA synthesis. From aawara from pontiff-playground.org Sat Aug 23 09:44:33 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Sat Aug 23 13:00:06 2008 Subject: KCl in RT-PCR References: Message-ID: In , sayeedansarmaricar@gmail.com wrote: > hi, can anyone tell me why do we use KCl in the reaction buffer for > cDNA synthesis. I don't know, but I can wager a guess. Much of the early biochemistry on reverse transcriptase was done on AMV reverse transcriptase (from avian myeloblastosis virus - a chicken retrovirus). The MLV enzyme was much harder to purify because it was harder to make large amounts of murine leukemia virus - this is in the pre-cloning & expression days. AMV RT exists in two forms - an alpha/beta heterodimer, and a beta/beta homodimer. Both these forms have polymerase and RnaseH activity - the beta/beta form has higher RnaseH activity. Anyway, polymerase processivity for AMV RT was slightly higher when K was the cation rather than Na, without affecting the RnaseH processivity. I worked on AMV RT in the 1980s, and it was my observation that this increased processivity with K was more easily observed with the beta/beta form than the alpha/beta form - so my guess is that the early preps used by Hurwitz and Spiegelman had more beta/beta RT than alpha/beta RT. I don't think this salt preference has any bearing on the recombinant "monomeric" RTs that are used commercially today - almost all of them are modified forms of MLV RT. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From dinshas209 from yahoo.co.uk Tue Aug 26 09:41:42 2008 From: dinshas209 from yahoo.co.uk (shashi sharma) Date: Tue Aug 26 13:34:57 2008 Subject: Transfection in MEF cell lines Message-ID: <368694.23235.qm@web25002.mail.ukl.yahoo.com> Dear all i am suffreing with a serious problem in transfection my drug product into the MEF cell lines. Kindly suggest some idea to it. with regards Sharma SK Send instant messages to your online friends http://uk.messenger.yahoo.com From R.Jayakumar from roswellpark.org Tue Aug 26 14:35:55 2008 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Tue Aug 26 18:27:09 2008 Subject: Transfection in MEF cell lines In-Reply-To: <368694.23235.qm@web25002.mail.ukl.yahoo.com> References: <368694.23235.qm@web25002.mail.ukl.yahoo.com> Message-ID: <97101976F8A044468CA74FE11883B90E173E7B01@VISTA.roswellpark.org> AND the problem??????? Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of shashi sharma Sent: Tuesday, August 26, 2008 10:42 AM To: methods@magpie.bio.indiana.edu Subject: Transfection in MEF cell lines Dear all i am suffreing with a serious problem in transfection my drug product into the MEF cell lines. Kindly suggest some idea to it. with regards Sharma SK Send instant messages to your online friends http://uk.messenger.yahoo.com _______________________________________________ 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 shauke from uni-bremen.de Wed Aug 27 03:30:29 2008 From: shauke from uni-bremen.de (Bluescript) Date: Wed Aug 27 12:30:40 2008 Subject: Counterstain for AEC AND BCIP/NBT Message-ID: 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. Has anybody a good idea? Thank you very much for your help in advance, Sven From engelbert_buxbaum from hotmail.com Wed Aug 27 13:53:17 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Thu Aug 28 11:03:47 2008 Subject: Protein Extraction from Fat References: Message-ID: Am 21.08.2008, 14:30 Uhr, schrieb Joel Schneider : > I am currently interested in analyzing the protein content of a fat > sample I > recently harvested. Is there a particular protein extraction method > recommended for fat? That depends on what type of protein (large/small, hydrophilic/hydrophobic) you want to extract for which application (IEF, MS, aa analysis, Edman sequencing...). It also depends on the nature of the sample, small amounts of protein in a dietary fat would certainly be treated differently than the membrane proteins from a cell homogenisation. In principle, you have two possibilities: - solubilising proteins and fat in detergents followed by separation of protein containing micelles - removing the fat with organic solvents followed by solubilisation of the protein residue (e.g. the Wessel & Fl?gge procedure). From engelbert_buxbaum from hotmail.com Wed Aug 27 14:10:34 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Thu Aug 28 11:03:57 2008 Subject: Counterstain for AEC AND BCIP/NBT References: Message-ID: 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. > Has anybody a good idea? Thank you very much for your help in advance, Intercalating DNA stains are commonly used to mark nuclei: bisbenzimide, DAPI, Propidium iodide and a host of proprietary dyes (check the Molecular Probes catalogue). From shauke from uni-bremen.de Thu Aug 28 02:06:09 2008 From: shauke from uni-bremen.de (Bluescript) Date: Thu Aug 28 11:04:13 2008 Subject: Counterstain for AEC AND BCIP/NBT References: Message-ID: 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. > > Has anybody a good idea? Thank you very much for your help in advance, > > Intercalating DNA stains are commonly used to mark nuclei: bisbenzimide, ? > DAPI, Propidium iodide and a host of proprietary dyes (check the Molecular ? > Probes catalogue). 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! From valerienathan9 from googlemail.com Thu Aug 28 05:33:39 2008 From: valerienathan9 from googlemail.com (valerienathan9@googlemail.com) Date: Thu Aug 28 11:04:55 2008 Subject: Qiagen LyseBlue References: Message-ID: <11822ff5-67c5-4654-b557-21b7ecb010e4@e39g2000hsf.googlegroups.com> On Jul 17, 9:51?pm, "Sharon Waldrop" wrote: > Hello Group, > Does anyone know what components are in LyseBlue? Do you, perchance, > know the formula? > SLW Try thymolphthalein 43 mg/ml ethanol. VN From rashmi1208 from googlemail.com Thu Aug 28 07:40:35 2008 From: rashmi1208 from googlemail.com (Rashmi Srivastava) Date: Thu Aug 28 15:29:40 2008 Subject: (no subject) Message-ID: <7a51f5da0808280540r718daceaqfa4a1a6b23d8d824@mail.gmail.com> Hi, Could someone please tell what's good about M15 cells? Thanks From vetdrrkc from gmail.com Fri Aug 29 11:00:40 2008 From: vetdrrkc from gmail.com (Ratan K) Date: Fri Aug 29 15:30:59 2008 Subject: RNA stability and sodium citrate buffer Message-ID: Hi I am a new subscriber to Methods Mailing List. Can any one suggest me that heating ethanol fixed cryosection with sodium citrate (10mM, pH 7.0) at 90 degree celcius for 15 min will degrade RNA present in cryosection. Thanks in advance. -- Cheers Ratan From Glen.Alberts from vai.org Fri Aug 29 14:14:48 2008 From: Glen.Alberts from vai.org (Alberts, Glen) Date: Fri Aug 29 15:31:15 2008 Subject: Red/ET recombination Message-ID: <9A6509A94A33124187515A9901DC551F09522CE3A9@VAIEXCH05.vai.org> Tam, Are you still working with the Red/ET kit? I found your old posting. We have tried the kit but have many challenges with it. Glen -Van Andel Institute (Michigan) This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient(s) please contact the sender by reply email and destroy all copies of the original message. Thank you. From stxsz1 from nottingham.ac.uk Fri Aug 29 17:33:15 2008 From: stxsz1 from nottingham.ac.uk (Zhong Silin) Date: Fri Aug 29 18:48:59 2008 Subject: phylogenetic tree server References: <200808291704.m7TH4kU19973@net.bio.net> Message-ID: Hi, If I have a conserved gene or a protein with a conserved domain, where can I view a phylogenetic tree of all the organisms containing its homologue? I have done this ages ago. Maybe in NCBI or InterPro. I remember it was just a few clicks and I can view and modify the tree on that website. But I just can't find it now. Sorry guys it is not a very challenging question SZ 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 vetdrrkc from gmail.com Sat Aug 30 21:44:28 2008 From: vetdrrkc from gmail.com (Ratan K) Date: Sun Aug 31 12:40:45 2008 Subject: Thanks Message-ID: DEAR DK THANK YOU VERY MUCH. I WONDER IF SUCH RNA COULD BE USED FOR AMPLIFICATION AND MICROARRAY LATER. ONCE AGAIN THANKS. REGARDS RATAN -- Cheers Ratan