From siheng.xiang from gmail.com Tue Apr 1 07:20:35 2008 From: siheng.xiang from gmail.com (kalva) Date: Tue Apr 1 11:50:20 2008 Subject: Transformation onto M9 Minimal Media References: Message-ID: <419a6804-a382-4c9c-82b3-2a6a3f927c90@i29g2000prf.googlegroups.com> have u tried add 0.5% -1% glucose in ur LB media (after autoclaved), which may reduce some leaky expression of T7? On 3=D4=C228=C8=D5, =C9=CF=CE=E76=CA=B130=B7=D6, cv2...@columbia.edu wrote: > Hello > I have a toxic protein expressed from a T7 promoter in Pet21 D. > > I'm trying to harvest the protein, but getting little yield. > > I was told to do everything in minimal media to reduce any leaky > expression of T7 > (I.e. to make certain there is no break down of lactose analogs found in L= B). > > I am using M8 MM + .4% glucose + 1mg/ML CAA + 100 ug/ML Ampicillin > > Unfortunately, I am having a hard time even TRANSFORMING my plasmids > into BL21(DE3)pLysS (there should be little expression in this strain > anyway) in Minimal Media. > > Even the puc 19 positive control failed. > > I did the typical 30 minutes on ice, 45 sec heat shock at 42C, 2 > minutes ice, 1 hour recovery (in M9 MM) . > > twice this has failed (it also failed once on E.coli). > > IS there any reason I should have such problems? > Please help if you can > > thanks > Christal From amanda.beltramini from gmail.com Tue Apr 1 13:40:53 2008 From: amanda.beltramini from gmail.com (Amanda Beltramini) Date: Tue Apr 1 21:02:42 2008 Subject: M9 Media Message-ID: In response to your minimal media question, have you tried using a richer media for recovery (SOC, LB, e.g.)? Since the protein is toxic, I would recommend doing this and perhaps extending recovery to 1.5 hours. Just something to try. From jma from ddt.biochem.umn.edu Tue Apr 1 18:37:06 2008 From: jma from ddt.biochem.umn.edu (Mike Autry) Date: Tue Apr 1 21:03:03 2008 Subject: Coated plates for fluorescence imaging of baculovirus-infected insect cells? Message-ID: Methods: Which coating is best to keep baculovirus-infected insect cells adhered to microscope plates: poly-lysine, integrin, laminin, etc? Any recommendations on plate type and vendor availability? I have a major problem doing FRET microscopy on Sf21 insect cells infected with recombinant AcNPV baculoviruses: following infection, the Sf21 cells detach from the glass microscope plate and roll around, especially when I change filter cubes (donor, acceptor, FRET dichroics) during photobleaching experiments. I am expressing ER membrane proteins tagged with cyan and yellow fluorescent proteins (CFP, YFP). Thanks, J. Michael Autry, PhD Research Associate, University of Minnesota Minnesota Muscle Laboratory, David D. Thomas, PI http://ddt.biochem.umn.edu/ From grand.david from gmail.com Wed Apr 2 03:50:15 2008 From: grand.david from gmail.com (David Grand) Date: Wed Apr 2 14:24:37 2008 Subject: RE-GST contamination of GST fusion protein Message-ID: <4ff842830804020150l51cb3315je75dc878186bcce7@mail.gmail.com> Hi Paul.Phelan Can you cleave your target from GST with a protease recognition site. Or, try to use reducing buffer when doing your gel filtration ... Reducing the GST dimer as monomer will let you separate it from your fusion. Don't know if for your application reducing buffer will be a limitation. And I have never tested it. Do a reducing test, try different DTT or B-mercapto concentrations, load it on a non reducing SDS-PAGE and find the best lower limit of [DTT or B mercapto] needed. Probably a strong reduction isn't good for your target protein so doing this test can be a good information. Treat your co-purified fraction with this buffer before gel filtration and see what happen. You will probably be able to separate them by size, as monomers after treatment... Doing denaturation is good but you will have problems for the renaturation as it's a difficult process. If someone can help :) Cheers. David. In , Paul J. Phelan wrote: > To whom it concerns, > This question maybe have been asked before, but I am having trouble > with separating GST from a preparation of GST-Importin alpha. I have > maybe 10-20% GST co-purifying with the fusion protein. The pI of both > proteins is the same, so ion exchange chromatography does not separate > them, and even on a Sephacryl S-100 gel filtration column, the two > proteins (87 kDa fusion, 27 kDa GST) eluted together. Does anyone have > a clever trick to remove GST from a GST fusion prep.? > My next attempt is going to be to try gel filtration again but with 0.3 > M NaCl and maybe some detergent to try to separate the proteins. > > Any advice would be greatly appreciated, thanks GST dimerizes, so it isn't surprising that the cleaved GST moiety co-purifies with your GST-Rch1 fusion. In my limited experience with GST fusions, I've never encountered this premature cleavage that you're seeing, so my suggestion below is not tested. Why don't you denature your GST/GST-Rch1 mixture, and then separate them by gel filtration? Using 3M GdHCl? AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From Andreas.Ploner from i-med.ac.at Wed Apr 2 06:36:48 2008 From: Andreas.Ploner from i-med.ac.at (Andreas Ploner) Date: Wed Apr 2 14:24:43 2008 Subject: LNA transfection! Message-ID: <47f36fd0$0$12642$3b214f66@aconews.univie.ac.at> Hi everyone! I've got a problem, maybe one of can help me. I want to transfect Locked nucleic acids (LNAs) into 293T cells. HAs anyone of you expereince with LNA transfection and whch transfection reagent do you use? Most publication use Lipofectamin. At the moment im using Metafectene (www.biontex.com). At the moment it seems that knock down somehow works, but I can't find out easily how efficient LNAs get transfected- so I'd need some transfection reagent to comapre it to! Thanks for your answers! Andi From stxsz1 from nottingham.ac.uk Wed Apr 2 17:19:58 2008 From: stxsz1 from nottingham.ac.uk (Zhong Silin) Date: Wed Apr 2 17:40:40 2008 Subject: Why use K+ and NH4+ in Taq buffer? Message-ID: Can someone explain to me what is the role of K+ and NH4+ in the PCR buffer? I noticed most of the suppliers are using the NH4 buffer. Only NEB sticks to the original KCl buffer. For example Bioline 1x Taq buffer: 16mM (NH4)2SO4, 67mM Tris NEB 1x Taq standard buffer: 50mM KCl, 10 mM Tris and why the Bioline use such a high concentration of Tris? Thanks in advance Silin 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 Wed Apr 2 18:02:11 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Apr 2 19:46:35 2008 Subject: Why use K+ and NH4+ in Taq buffer? References: Message-ID: Dear SiLin, total ionic strength doesn't differ so much. It's most likely due to patent and other legal issues and/or due to the composition of the Taq storage buffer that the PCR buffers differ in composition. Seems that in real life these differences do not affect Taq much but of course require you to optimise a PCR for each polymerase/buffer composition. (another way nasty companies annoy scientists to stay with their products). Furthermore, (that's my personal explanation for the ammonium salts) some Taqs are hot start and reversibly blocked (by imino group formation with eg lysines) with formaldehyde (btw, thats how some people make their homebrew hot start Taq, too). This formaldehyde - once liberated by heat- can be caught by the excess of ammonium ions and thus won't afftect the Taq anymore. Best regards, Wo From yansun2005 from gmail.com Sun Apr 6 19:15:14 2008 From: yansun2005 from gmail.com (yan sun) Date: Mon Apr 7 12:32:39 2008 Subject: PAGE purification and analysis Message-ID: Dear all, I was confused by my PAGE gel purification for a while. I tried to purified spin labeled DNA and non labeled DNA through 20% polyacrylamide gel. My DNA is around 8,200 dalton, the attached spin label material MW is around 200g/mole, which means I try to separate two DNA bands, the molecular weight difference is 200 dalton. My DNA is 27 mer. The problem is that two bands are overlapped. the band isn't sharp, the resolution is not good, the band is in meniscus shape. wasn't straight at all:( My lab mate who successfully purified this two band get very sharp and clear bands.I "exactly" follow her protocol. but do not get the satisfied result:( I used constant power, 15w. my guess is the temperature is too high, should I run the gel in the cold room? but I pretty sure she run it in RT. I pour 25ml acrylamide solution, should I degas it first? my 10% APS is new, TEMED is good. is that because the poor gel quality? but she didn't degas the gel. I know it really depends on individual hand-on experience. I don't really know how to improve it, how to get sharp and clear bands? Anybody has suggestion? Thx a lot! -- Y. F.S. From adrien.vetterli from helsinki.fi Mon Apr 7 00:49:16 2008 From: adrien.vetterli from helsinki.fi (adrien) Date: Mon Apr 7 12:32:45 2008 Subject: Direct Sequencing of degenerated PCR products Message-ID: <47F9B5DC.5000404@helsinki.fi> Hi everybody, I would like to sequence PCR products obtained from a reaction with degenerated primers without having to go through the cloning thing. The primers to be used in the sequencing reaction are degenerated as well. Has anyone tried that who can tell me if the results are usable ? I know of some scientists who have designed degenerated primers with SP6 or T7 extensions and use these sites during sequencing instead of the degenerated ones but I am not going there either. thank you for your help. adrien From tk from mit.edu Mon Apr 7 22:42:08 2008 From: tk from mit.edu (Tom Knight) Date: Tue Apr 8 15:25:49 2008 Subject: Direct Sequencing of degenerated PCR products References: Message-ID: adrien writes: > I would like to sequence PCR products obtained from a reaction with > degenerated primers without having to go through the cloning > thing. The primers to be used in the sequencing reaction are > degenerated as well. Has anyone tried that who can tell me if the > results are usable ? I know of some scientists who have designed > degenerated primers with SP6 or T7 extensions and use these sites > during sequencing instead of the degenerated ones but I am not going > there either. I've done this with no special considerations. Just gel isolation of the PCR product band and addition of one of the degenerate primers. You can't determine the specific bases of the degenerate primers you have used by sequencing in the opposite direction -- they give mixed or incorrect results for the degenerate positions. From ivanoov from gmail.com Tue Apr 8 04:28:50 2008 From: ivanoov from gmail.com (chovek69) Date: Tue Apr 8 15:26:17 2008 Subject: Why use K+ and NH4+ in Taq buffer? References: Message-ID: On Apr 3, 2:02?am, WS wrote: > Dear SiLin, > > total ionic strength doesn't differ so much. It's most likely due to > patent and other legal issues and/or due to the composition of the Taq > storage buffer that the PCR buffers differ in composition. Seems that > in real life these differences do not affect Taq ?much but of course > require you to optimise a PCR for each polymerase/buffer composition. > (another way nasty companies annoy scientists to stay with their > products). > > Furthermore, (that's my personal explanation for the ammonium salts) > some Taqs are hot start and reversibly blocked (by imino group > formation with eg lysines) with formaldehyde (btw, thats how some > people make their homebrew hot start Taq, too). This formaldehyde - > once liberated by heat- can be caught by the excess of ammonium ions > and thus won't afftect the Taq anymore. > > Best regards, > > Wo Hi, Check the bible http://info.med.yale.edu/genetics/ward/tavi/p06.html http://info.med.yale.edu/genetics/ward/tavi/p07.html check also the references From my own experience I can say that KCl-based buffers are far superior than (NH4)2SO4-based ones in terms of PCR sensitivity, and specificity (maybe because the latter ones require far more Mg2+ which leads to unspecific amplifications) From engelbert_buxbaum from hotmail.com Tue Apr 8 12:41:20 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Apr 8 15:26:23 2008 Subject: PAGE purification and analysis References: Message-ID: Am 06.04.2008, 20:15 Uhr, schrieb yan sun : > Dear all, > I was confused by my PAGE gel purification for a while. > I tried to purified spin labeled DNA and non labeled DNA through 20% > polyacrylamide gel. My DNA is around 8,200 dalton, the attached spin > label > material MW is around 200g/mole, which means I try to separate two DNA > bands, the molecular weight difference is 200 dalton. I do PAGE for proteins rather than DNA, but perhaps some hints: - The MW difference is very small, only about 3%. Try optimizing the separation by using gradient gels and by using a gel concentration where your molecule ends up 2/3 - 3/4 down the gel. - If you can find conditions where labeled and unlabeled DNA have different net charge that would greatly improve separation. Without knowing exactly what label you attach where, it is difficult to give specifc advice. From mfarizab from unal.edu.co Tue Apr 8 18:03:46 2008 From: mfarizab from unal.edu.co (Manuel Fernando Ariza Botero) Date: Tue Apr 8 19:29:54 2008 Subject: PAGE purification and analysis (yan sun) In-Reply-To: <200804081704.m38H4IT18950@net.bio.net> References: <200804081704.m38H4IT18950@net.bio.net> Message-ID: What kind of loading buffer are you using? Why is the PAGE concentration so high? Fernando Ariza ----- Mensaje original ----- De: methods-request@oat.bio.indiana.edu Fecha: Martes, Abril 8, 2008 12:04 pm Asunto: Methods Digest, Vol 35, Issue 4 > 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. Direct Sequencing of degenerated PCR products (adrien) > 2. PAGE purification and analysis (yan sun) > > > ------------------------------------------------------------------- > --- > > Message: 1 > Date: Mon, 07 Apr 2008 08:49:16 +0300 > From: adrien > Subject: Direct Sequencing of degenerated PCR products > To: methods@magpie.bio.indiana.edu > Message-ID: <47F9B5DC.5000404@helsinki.fi> > Content-Type: text/plain; charset="iso-8859-1" > > Hi everybody, > > I would like to sequence PCR products obtained from a reaction > with > degenerated primers without having to go through the cloning > thing. The > primers to be used in the sequencing reaction are degenerated as > well. > Has anyone tried that who can tell me if the results are usable ? > I know > of some scientists who have designed degenerated primers with SP6 > or T7 > extensions and use these sites during sequencing instead of the > degenerated ones but I am not going there either. > > thank you for your help. > > adrien > > > ------------------------------ > > Message: 2 > Date: Sun, 6 Apr 2008 20:15:14 -0400 > From: "yan sun" > Subject: PAGE purification and analysis > To: Methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Dear all, > I was confused by my PAGE gel purification for a while. > I tried to purified spin labeled DNA and non labeled DNA through 20% > polyacrylamide gel. My DNA is around 8,200 dalton, the attached > spin label > material MW is around 200g/mole, which means I try to separate two DNA > bands, the molecular weight difference is 200 dalton. My DNA is 27 > mer.The problem is that two bands are overlapped. the band isn't > sharp, the > resolution is not good, the band is in meniscus shape. wasn't > straight at > all:( > My lab mate who successfully purified this two band get very > sharp and > clear bands.I "exactly" follow her protocol. but do not get the > satisfiedresult:( > I used constant power, 15w. > my guess is the temperature is too high, should I run the gel in > the cold > room? but I pretty sure she run it in RT. > I pour 25ml acrylamide solution, should I degas it first? my 10% > APS is new, > TEMED is good. is that because the poor gel quality? > but she didn't degas the gel. I know it really depends on > individual hand-on > experience. > I don't really know how to improve it, how to get sharp and clear > bands?Anybody has suggestion? > Thx a lot! > > -- > Y. F.S. > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 35, Issue 4 > ************************************** > From iayork from panix.com Thu Apr 10 12:26:03 2008 From: iayork from panix.com (Ian A. York) Date: Thu Apr 10 19:07:19 2008 Subject: Cells spontaneously zeocin resistant? Message-ID: We've routinely made stable cell lines using plasmids containing zeocin resistance. However, recently some of our starting cell lines have apparently become spontaneously resistant to zeocin at fairly high doses, at least 1000 ug/ml. We've considered a couple of possible explanations. (1) Have we mixed up cell lines so that we're accidentally growing a resistant stable cell line? If so none of the markers that go in with the resistance are present, including GFP (the resistance gene we use is from Invitrogens pTracerCMV2 plasmids, which contains a GFP/ZeoR fusion protein). We have also gone back to our earliest freezes from liquid nitrogen without success. And at least two rather different cell lines are behaving this way, even though the two lines look and behave differently otherwise. (2) Has our zeocin gone bad? It's relatively fresh and as far as I can tell has been treated properly (i.e.e kept frozen). It kills some other cell lines as expected at reasonable concentrations, 100 ug/ml. I am reluctant to spring for a new batch of zeocin if this is still good, because it's rather expensive. Are there other explanations? Could mycoplasma contamination cause zeocin resistance? Thanks, Ian -- Ian York (iayork@panix.com) "-but as he was a York, I am rather inclined to suppose him a very respectable Man." -Jane Austen, The History of England From iayork from panix.com Fri Apr 11 10:29:18 2008 From: iayork from panix.com (Ian A. York) Date: Fri Apr 11 12:52:50 2008 Subject: Cells spontaneously zeocin resistant? References: Message-ID: In article , DK wrote: >In article , iayork@panix.com (Ian >A. York) wrote: >>We've routinely made stable cell lines using plasmids containing >>zeocin resistance. However, recently some of our starting cell lines >>have apparently become spontaneously resistant to zeocin at fairly >>high doses, at least 1000 ug/ml. >> >The whole cell lines became resistant or you start occasionally seeing >clones in the mock transformations? Either the whole cell line, or at least a large fraction of the cells. A well with zeo, seeded at about 10% confluence, becomes confluent at roughly the same rate as an untreated well. Ian -- Ian York (iayork@panix.com) "-but as he was a York, I am rather inclined to suppose him a very respectable Man." -Jane Austen, The History of England From agbiok4 from gmail.com Tue Apr 15 03:34:19 2008 From: agbiok4 from gmail.com (kamalaker nasani) Date: Tue Apr 15 12:26:37 2008 Subject: good morning Message-ID: I have a small doubt regarding Southern blot. After blotting membrane and x ray film we will keep them in -70 degrees to develop image of the membrane. Can any one tell the logic behind this process. If anybody have the protocol for this technique please share with me. Hope to c reply soon. -- 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 nick_theodorakis from hotmail.com Tue Apr 15 21:45:23 2008 From: nick_theodorakis from hotmail.com (Nick Theodorakis) Date: Wed Apr 16 12:54:28 2008 Subject: good morning In-Reply-To: References: Message-ID: DK wrote: > In article , "kamalaker nasani" wrote: >> I have a small doubt regarding Southern blot. >> >> After blotting membrane and x ray film we will keep them in -70 degrees to >> develop image of the membrane. Can any one tell the logic behind this >> process. > > The way it was explained to me long ago made perfect sense > so I never questioned it despite knowing next to nothing about > chemistry of photography. Here it is: > > It takes more than one hit to the same atom to convert Ag+ into > elementary silver with photons. The first hit/step is chemically > reversible - excited Ag+* can spontaneously relax back to Ag+. > In other words, it's a chemical reaction and therefore depends on > temperature. The second is not - once Ag0 is formed, it's a done > deal. That is why in old days it was recommended to "preflash" > X-ray film for maximum sensitivity. So in your case the lower > temperature "freezes" the reverse reaction, thereby increasing > sensitivity. > > DK You are essentially correct, but I want to add that implicit in your description is the assumption that one is using a light-based exposure method such as using an intensifying screen. If you are only using betas to expose, you don't need to cool the film. The phenomenon is called "reciprocity failure," in case anyone wants to google to learn more about it. It also used to be a problem for other long light exposures such as astrophotography. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From tfitzwater from somalogic.com Wed Apr 16 09:21:08 2008 From: tfitzwater from somalogic.com (Tim Fitzwater) Date: Wed Apr 16 12:54:55 2008 Subject: good morning Message-ID: <771BF0D090CE73449F7495D31E0BA0D1217D96@ELM.sladmin.com> Because an old Amersham Guide to Autoradiography contains the best explanation of pre-flashing that I've ever found, it seems simplest to generate a Reader's Digest version: "Photographic emulsion consists of silver halide crystals suspended in a clear phase composed mainly of gelatin. When a beta particle or gamma ray passes through such an emulsion the silver halide crystals respond, with each emission converting several silver ions to silver atoms." "These crystals constitute the grains of the film. The silver bromide forms a regular lattice, however within each crystal there will be a number of faults. When a photon of light enters the crystal, there is a high probability that it will give up its energy to one of the orbital electrons in the lattice. When such an electron has acquired enough energy it will leave its orbit finally stopping at a fault. Here, a silver ion accepts the electron to become an atom of metallic silver. This silver constitutes the latent image." "To produce a developable image each silver halide crystal requires several photons of visible light (approximately 5 in average emulsions), each of which produces an atom of metallic silver within the crystal." "By comparison, when a beta particle or gamma ray passes through a photographic emulsion, energy is lost in a series of interactions with orbital electrons in the silver halide crystals. This results in silver deposition at a number of faults, again forming a latent image." "A single hit by a beta particle or gamma ray can produce hundreds of silver atoms from silver halide crystals in film emulsion, but a single photon of light from an intensifying screen produces only a single silver atom. Although two or more silver atoms in a silver halide crystal are stable, a single silver atom is unstable and reverts to a silver ion very rapidly." "This means that the probability of a second photon being captured before the First silver atom has reverted is greater for larger amount of radioactivity than for small amounts. Hence small amounts of radioactivity are under-represented for both fluorography and intensifying screens." "This problem can be completely overcome by a combination of pre-flashing the film and exposing at -70?C." -- Amersham "Pre-exposure to an instantaneous flash of light overcomes the problem of low intensities of light producing disproportionately faint images. It does so by providing many of the halide crystals with a stable pair of silver atoms. Sensitivity is increased, allowing quantification of images because all photons contribute equally to the image on pre-flashed film." "A linear response of the film to light produced from the radioactivity in the sample is obtained when the film has been pre-flashed sufficiently to increase absorbance of 0.1 to 0.2 (A540) absorbance units above that of unexposed developed film. A further increase in the fog level above 0.2 reverses the deviation from linearity. Thus a pre-flash of too much light causes over-representation of small amounts of radioactivity." "Lowering the temperature to -70?C increases the stability of a single silver atom. The reversion to a silver ion is slowed, increasing the time available to capture a second photon and thus produce a stable pair of silver atoms." "Both pre-flashing and exposure at -70?C increase sensitivity on their own. Only the combination of the two completely corrects the response of the film, giving a linear response to the amount of activity in the sample." --Amersham Pre-flashing is essential if you want accurate densitometry data. The 14C calibration strip (American Radiolabeled Chemicals, Inc. catalog # ARC-146) will allow you to determine the linear range of the film for each exposure and convert scan values to ?Ci. This exposure strip can also be used to quantitate phosphorimager values as ?Ci rather than arbitrary units. Densitometer and Phosphorimager can be calibrated for each exposure to directly convert to ?Ci values. While not linear, temperature can be used to modulate exposure times when using a Lightning Plus intensifying screen. If a -70?C exposure requires 1 hour, -20?C requires about 2 hours, 4?C requires about 3.5 hours and room temperature requires about 7 hours exposure. This allows you to find an exposure that fits your schedule. Note that if time in the freezer is too short the film won't have time to get cold. The Lightning Plus intensifying screen has almost no effect at room temperature, so it does not need to be removed from the cassette. A sheet of aluminum foil covering intensifying screens that are adhered to the cassette eliminates any effect by blocking screen response. From analia_alet from intech.gov.ar Thu Apr 17 08:21:23 2008 From: analia_alet from intech.gov.ar (Analia Alet) Date: Thu Apr 17 12:21:48 2008 Subject: good morning In-Reply-To: <200804161705.m3GH51T28460@net.bio.net> References: <200804161705.m3GH51T28460@net.bio.net> Message-ID: <1208438483.48074ed317779@webmail.intech.gov.ar> Another reason for this is to improve southern quality. at high temperatures, the electrons impact very fast in the x-ray film, making a very large spot, so you don't have "a band" rader it is a big spot without boundaries. This is the case for P32. But if you are using C (not in southern blot but in other techniques) you don?t need to place the [x-ray films+membrane] at -70?C because de decay constant for this isotope is very low, so you just leave your films at room temperature for almost a week. At least this is how they explained this to me best regards Anal?a Mensaje citado por methods-request@oat.bio.indiana.edu: > 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..." > In article , "kamalaker nasani" wrote: >I have a small doubt regarding Southern blot. > >After blotting membrane and x ray film we will keep them in -70 degrees to >develop image of the membrane. Can any one tell the logic behind this >process. The way it was explained to me long ago made perfect sense so I never questioned it despite knowing next to nothing about chemistry of photography. Here it is: It takes more than one hit to the same atom to convert Ag+ into elementary silver with photons. The first hit/step is chemically reversible - excited Ag+* can spontaneously relax back to Ag+. In other words, it's a chemical reaction and therefore depends on temperature. The second is not - once Ag0 is formed, it's a done deal. That is why in old days it was recommended to "preflash" X-ray film for maximum sensitivity. So in your case the lower temperature "freezes" the reverse reaction, thereby increasing sensitivity. DK From realpeqm from niaid.nih.gov Thu Apr 17 10:59:07 2008 From: realpeqm from niaid.nih.gov (Realpe-Quintero, Mauricio (NIH/NIAID) [F]) Date: Thu Apr 17 12:21:55 2008 Subject: FLAC METHOD TO OBTAIN SEQUENCE INFO FROM RNA VIRUSES Message-ID: <0FEED6FB2F3CF24384F2711FD61CC00208EFD3@NIHCESMLBX16.nih.gov> Hi !!. I am planning to run a couple of reactions based on the FLAC method and decided to ask the list members about theirs experiences. Just trying to figure out how much possible would be to avoid the hassle of cloning sequencing and go directly to the ABI, instead. Any input would be highly appreciated, thanks in advance, Mauricio Realpe (LID/NIAID/NIH) From ssokolow from swing.be Mon Apr 21 12:18:00 2008 From: ssokolow from swing.be (Sophie) Date: Mon Apr 21 12:31:18 2008 Subject: Guanidine and SDS-page gel Message-ID: <480CCC48.9060703@swing.be> I read on this forum that it is possible to run guanidine samples if they are diluted. Which final concentration of guanidine do you recommend to avoid SDS precipitation? Thanks, Sophie From ben.long from yourfinger.anu.edu.au Mon Apr 21 18:04:29 2008 From: ben.long from yourfinger.anu.edu.au (Bean Long) Date: Mon Apr 21 18:32:20 2008 Subject: Guanidine and SDS-page gel In-Reply-To: References: Message-ID: <480d1d7e@clarion.carno.net.au> Sophie wrote: > I read on this forum that it is possible to run guanidine samples if > they are diluted. Which final concentration of guanidine do you > recommend to avoid SDS precipitation? > Thanks, > Sophie I'd recommend urea instead if possible. 8M urea in samples is not a problem for SDS-PAGE. Unfortunately I learned this a long time ago after a dreadful experience with guanidine and now can't recall the lowest reasonable concentration for loading. Best of luck. From matti.savoila from gmail.com Tue Apr 22 04:38:37 2008 From: matti.savoila from gmail.com (Matti Savoila) Date: Tue Apr 22 08:43:22 2008 Subject: Primer analysis software. Message-ID: <2cf61c0f0804220238l210134e0rb864224199074610@mail.gmail.com> Hello everyone! I'm losing my virginity on mailing to the methods list here and now. My question is about, preferably web-based, primer analysis software? Is there some to be recommended? My main concern is primer-dimers, I'm running real-time PCR with hydrolysis probes, with single primers+probe per capillary. I have used something on Sigma's pages, but I am unable to find it anymore. Have used quite some time to google around, but to no avail, can someone recommend some good software? Best regards Matti Savoila From mayte from titan.ipicyt.edu.mx Tue Apr 22 16:45:35 2008 From: mayte from titan.ipicyt.edu.mx (MAYTE GUADALUPE CERVANTES BADILLO) Date: Tue Apr 22 21:42:11 2008 Subject: primer analysis software (Savoila) In-Reply-To: <200804221705.m3MH5jT06908@net.bio.net> References: <200804221705.m3MH5jT06908@net.bio.net> Message-ID: <20080422213221.M33079@titan.ipicyt.edu.mx> Message: 4 Date: Tue, 22 Apr 2008 12:38:37 +0300 From: "Matti Savoila" Subject: Primer analysis software. To: methods@magpie.bio.indiana.edu Message-ID: <2cf61c0f0804220238l210134e0rb864224199074610@mail.gmail.com> Content-Type: text/plain; charset=ISO-8859-1 Hello everyone! I'm losing my virginity on mailing to the methods list here and now. My question is about, preferably web-based, primer analysis software? Is there some to be recommended? My main concern is primer-dimers, I'm running real-time PCR with hydrolysis probes, with single primers+probe per capillary. I have used something on Sigma's pages, but I am unable to find it anymore. Have used quite some time to google around, but to no avail, can someone recommend some good software? Best regards Matti Savoila ------------------------------ Hi! There is a software, Fast PCR, that is easy to use. You can find it in this website: http://www.biocenter.helsinki.fi/bi/Programs/fastpcr.htm It has a manual and the information necessary for its use (PCR in silico, primers analysis, etc.) Good luck! CBM _____________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From tk from mit.edu Tue Apr 22 18:06:58 2008 From: tk from mit.edu (Tom Knight) Date: Tue Apr 22 21:42:18 2008 Subject: Primer analysis software. References: Message-ID: "Matti Savoila" writes: > My question is about, preferably web-based, primer analysis software? > Is there some to be recommended? My main concern is primer-dimers, I'm > running real-time PCR with hydrolysis probes, with single > primers+probe per capillary. Check out the IDT web page here: http://www.idtdna.com/SciTools/SciTools.aspx From mrinalsingha from gmail.com Wed Apr 23 02:37:39 2008 From: mrinalsingha from gmail.com (Mrinal Singha) Date: Wed Apr 23 11:51:15 2008 Subject: methods of breaking disulphide bonds Message-ID: <3e2824ee0804230037t17b86f86ycc370c3ac1b67670@mail.gmail.com> Respected Sir/Madam I am trying to form methyl ester of 4-Mercapto benzoic acid by using thionyl chloride and then adding methanol at room temparature. Although ester is formed but it is also forming disulfide. I want to cleave disulfide bond without cleavage of ester. Please tell me some suitable methods for this. Thank You Mrinal singha Student of Master of Science( Medicinal Chemistry) National Institute of Pharmaceutical Education and Research(NIPER) Punjab,India From matti.savoila from gmail.com Wed Apr 23 06:47:05 2008 From: matti.savoila from gmail.com (Matti Savoila) Date: Wed Apr 23 11:51:35 2008 Subject: Real time PCR. Funky fluorescence? Message-ID: <2cf61c0f0804230447n4136feb5wb8f51c8a07f7b8b3@mail.gmail.com> Hello everyone! I am doing some real time PCR with the LightCycler 1.5 instrument, but I'm getting weird results. The fluorescence rises, like it should, but suddenly in one cycle it plummets down. Examples: http://www.mv.helsinki.fi/savoila/d_kuvat/Nayte_4_edit.jpg http://www.mv.helsinki.fi/savoila/d_kuvat/Nayte_6_edit.jpg Have you seen behaviour like this, and more importantly, have you seen a solution to problems like this? Number 10 in both samples has been amplified normally, which is strange. If somebody has some ideas, I'd be thankful. Best regards Matti Savoila From pdeitik from bcm.tmc.edu Wed Apr 23 12:26:24 2008 From: pdeitik from bcm.tmc.edu (Deitiker, Philip R) Date: Wed Apr 23 14:39:26 2008 Subject: methods of breaking disulphide bonds In-Reply-To: <3e2824ee0804230037t17b86f86ycc370c3ac1b67670@mail.gmail.com> Message-ID: Disulfide bonds form as a result of oxidation. If oxygen is present in your solution during the reaction disulfides will slowly form. Oxygen, particularly in the presence of other factors forms hydroxide radicals, or ozone can form radicals that react with disulfides more rapidly. To prevent sulfhydryl groups from dimerizing try evacuating solutions that have been heated up to remove oxygen, or displace the oxygen by bubbling nitrogen, and evacuating, bubbling more nitrogen gas, and evacuating, several times. This will remove oxygen from the reaction. All solutions should be oxygen free. This will Reduce ("pun intended") the oxidation of sulfides. You might also want to look up to see what common biproducts of the reaction are on pubmed or the internet. There may be specific sulfides that are oxygen scavengers that are less reactive with thiols. Good luck. -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Mrinal Singha Sent: Wednesday, April 23, 2008 2:38 AM To: methods@magpie.bio.indiana.edu Subject: methods of breaking disulphide bonds Respected Sir/Madam I am trying to form methyl ester of 4-Mercapto benzoic acid by using thionyl chloride and then adding methanol at room temparature. Although ester is formed but it is also forming disulfide. I want to cleave disulfide bond without cleavage of ester. Please tell me some suitable methods for this. Thank You Mrinal singha Student of Master of Science( Medicinal Chemistry) National Institute of Pharmaceutical Education and Research(NIPER) Punjab,India _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From L.Kamalian from liverpool.ac.uk Thu Apr 24 04:13:06 2008 From: L.Kamalian from liverpool.ac.uk (Kamalian, Laleh) Date: Thu Apr 24 11:40:21 2008 Subject: Does Lipofectamine cause gene knock down? Message-ID: <31DC2DFFD70C174B8E3072803F894F91046C71@EVSSTAFF1.livad.liv.ac.uk> Hi everyone I have come across this problem. I have stably transfected my cells using Lipofectamine 2000 as the transfection reagent. So far, I have been using my parental cells as the calibrator during transient transfection. Now for the stable transfection I have used scrambled si RNA oligonucleotide to transfect my cells and used a pool of these cells as my control. Using quantitative PCR to measure the mRNA of the colony cells and the control cells, my colony cells show about 70% gene knock down comparing to the scrambled si RNA control cells, which is good. However comparing to the parental cells, the control shows about 90% less expression of the gene of interest. Once before during my transient transfection I had the same problem when used "no DNA" as my negative control i.e. Lipofectamine without any DNA. At that time I had used western blot to measure the protein instead of QPCR. I had noticed that my negative control lysate had considerable decrease in protein expression. My question now is can Lipofectamine or transfection condition cause the gene knock down? It especially doesn't make sense now that my scrambled RNA control cells have been growing in the selective medial for at least 4 weeks now. I am thinking that even if the transfection condition had affected the cells transiently, it can't effect the mRNA level after being eliminated from the growth media. Am I right or am I missing something in here? Laleh. From kgilbride from shire.com Thu Apr 24 09:10:56 2008 From: kgilbride from shire.com (Gilbride, Kevin) Date: Thu Apr 24 11:40:46 2008 Subject: Real time PCR. Funky fluorescence? In-Reply-To: <2cf61c0f0804230447n4136feb5wb8f51c8a07f7b8b3@mail.gmail.com> Message-ID: <0F2CFCE6FC067A42813DFA1F9BB0697C010255EE@SHCHBEX03.corp.shire.com> I saw this once before while using ABI7700 and it turned out to be "falling Rox". The reference dye for some unknown reason loses its fluorescence and without the reference the target signal plummets. JMHO Good luck, K -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Matti Savoila Sent: Wednesday, April 23, 2008 7:47 AM To: methods@magpie.bio.indiana.edu Subject: Real time PCR. Funky fluorescence? Hello everyone! I am doing some real time PCR with the LightCycler 1.5 instrument, but I'm getting weird results. The fluorescence rises, like it should, but suddenly in one cycle it plummets down. Examples: http://www.mv.helsinki.fi/savoila/d_kuvat/Nayte_4_edit.jpg http://www.mv.helsinki.fi/savoila/d_kuvat/Nayte_6_edit.jpg Have you seen behaviour like this, and more importantly, have you seen a solution to problems like this? Number 10 in both samples has been amplified normally, which is strange. If somebody has some ideas, I'd be thankful. Best regards Matti Savoila _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods ______________________________________________________________________ This email has been scanned by the MessageLabs Email Security System. For more information please visit http://www.messagelabs.com/email ______________________________________________________________________ Please consider the environment before printing this e-mail This email and any files transmitted with it are confidential and may be legally privileged and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient please note that any disclosure, distribution, or copying of this email is strictly prohibited and may be unlawful. If received in error, please delete this email and any attachments and confirm this to the sender. www.shire.com From editor from gene-quantification.info Thu Apr 24 08:52:14 2008 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Thu Apr 24 11:44:00 2008 Subject: qPCR Newsletter - April 2008 Message-ID: qPCR Newsletter - April 2008 Dear researcher, dear Gene Quantification page reader, Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene Quantification homepage. The focus of this newsletter issue is: - update - High Resolution Melt (HRM) - update - quantification via real-time PCR - qPCR Symposium 2008 - European Seminar tour - qPCR application workshops in 2008 -------------------------------------------------------------------------------- High Resolution Melting (HRM) http://HRM.gene-quantification.info HRM is a novel, homogeneous, close-tube, post-PCR method, enabling genomic researchers to analyze genetic variations (SNPs, mutations, methylations) in PCR amplicons. It goes beyond the power of classical melting curve analysis by allowing to study the thermal denaturation of a double-stranded DNA in much more detail and with much higher information yield than ever before. HRM characterizes nucleic acid samples based on their disassociation (melting) behavior. Samples can be discriminated according to their sequence, length, GC content or strand complementarity. Even single base changes such as SNPs (single nucleotide polymorphisms) can be readily identified. The most important High Resolution Melting application is gene scanning - the search for the presence of unknown variations in PCR amplicons prior to or as an alternative to sequencing. Mutations in PCR products are detectable by High Resolution Melting because they change the shape of DNA melting curves. A combination of new- generation DNA dyes, high-end instrumentation and sophisticated analysis software allows to detect these changes and to derive information about the underlying sequence constellation. UPDATE - A lot new publications were added to the HRM pages => http://HRM.gene-quantification.info/ -------------------------------------------------------------------------------- real-time PCR - A technique enabling fast, quantitative and reliable results http://qPCR.gene-quantification.info Some of the limitations of end-point detection in PCR have been assuaged in real-time PCR systems, various are now on the market. These systems offer many general technical advantages, including reduced probabilities of variability and contamination, as well as online monitoring and the lack of need for post reaction analyses. Further, some of these systems were developed with contemporary applications such as quantitative PCR, multiplexing, and high- throughput analysis in mind. UPDATE - Alot of basic papers are presented and new publicatiojns were added to this section => http://qpcr.gene-quantification.info/ -------------------------------------------------------------------------------- qPCR Symposium USA 10. - 13. November 2008 Clarion Hotel San Francisco Airport, Millbrae, CA , USA http://www.gene-quantification.de/meetings.html#qpcr_usa High throughput platforms High throughput applications, real-time RT-PCR arrays, digital PCR Forthcoming technologies Immuno PCR, Methylation sensitive PCR, SNP analysis, High resolution melt, microRNA detection, Multiplex technologies Single-cell qPCR Pre-amplification, sub-cellular PCR, Expression heterogeneity, laser microdissection, FACS sorting, Enrichment of rare cells Multimarker diagnostics Disease markers, Tissue specific markers, Cancer markers, Stem cells, Differentiation markers, Cancer stem cells Real-time PCR Expression Profiling multivariate and multiway expression profiling, temporal expression profiling, spatiotemporal maps Pre-analytical Steps Sampling technologies, Extraction methods, Reverse Transcription, Quality Control, Standards, Standard Operating Procedures, Interlaboratory Exercises Normalization & Standardization Normalization strategies, Reference genes, Spikes, Standard curves,multiplexing, inter-run calibrators, quantification strategies, mRNA degradation Data management and data treatment software applications, data mining, data visualization, biostatistics, multivariate statistics Download TOPICS Flyer => http://www.gene-quantification.de/qPCR-Symposium-USA-2008-topics.pdf Download SPEAKERS Flyer => http://www.gene-quantification.de/qPCR-Symposium-USA-2008-speakers.pdf -------------------------------------------------------------------------------- With the new qPCR INFO PORTAL and all the presented tools we will help you with to find the right information about qPCR and related topics in Molecular Biology in the literature and in the World Wide Web. => Papers / Protocols / Methods / Databases / Alets / Feeds / Books / Forums / E-mail / Directory http://infoportal.gene-quantification.info/ -------------------------------------------------------------------------------- Upcoming Events World-wide academic and commercial qPCR Events http://events.gene-quantification.info/ Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars, qPCR Education Program, ... Please submit your qPCR event here => events@gene- quantification.info -------------------------------------------------------------------------------- WORKSHOP TATAA Biocenter Germany - qPCR Application workshops At the TATAA Biocenter Germany we offer qPCR application workshops, the 3-day Core Module and a 2-day Biostatistics Module. qPCR courses are held in regularly in G?teborg, Sweden, in English and in Freising- Weihenstephan, Germany, in German and English, and in Prague, Czech Republic in English and Czech. Depending on the occasion the workshop language and the different prices may apply. Further customized workshops and specialized trainings will be held as well across Europe and world-wide. TATAA Biocenter Germany courses are held in cooperation with the Institute of Physiology, located at the Technical University of Munich, in Freising-Weihenstephan, near Munich, very close to the Munich Airport (MUC). For more information and to register for the qPCR application workshops, please see our web page: http://tataa.gene-quantification.info/ Course Occasions 2008: 3-day qPCR Core Module (Mon. - Wed.) and 2-day BioStatistics Module (Thu. - Fri.) * 5 - 9th May 2008 (in Freising, Germany, Kurs wird in DEUTSCH gehalten, German language) * 7 - 11th July 2008 (in Freising, Germany, English language) Please register here => http://www.tataa.com/Courses/Courses.html -------------------------------------------------------------------------------- Forward Please send the qPCR NEWS to further scientists and friends who are interested in qPCR ! Best regards, Michael W. Pfaffl responsible Editor of the Gene Quantification Pages http://www.gene-quantification.info -------------------------------------------------------------------------------- If this newsletter is not displayed correctly by your email client, please use following LINK: http://qPCRnews.gene-quantification.info/ The qPCR NEWS and the Gene Quantification Pages are educational sites with the only purpose of facilitating access to qPCR related information on the internet. The qPCR NEWS and the Gene Quantification Pages are edited by Michael W. Pfaffl and powered by BioScience Events. Copyright ? 2005 - 2008 All rights reserved. Any unauthorized use, reproduction, or transfer of this message or its contents, in any medium, is strictly prohibited. Disclaimer & Copyrights are displayed on the homepage www.gene-quantification.com To subscribe or change your e-mail address in qPCR NEWS, and if you would like to receive future issues FREE of charge, please send an e- mail with the subject SUBSCRIBE to mailto:newsletter@gene- quantification.info?subject=SUBSCRIBE From arnec from bio.umass.edu Fri Apr 25 15:06:07 2008 From: arnec from bio.umass.edu (Arne K Christensen) Date: Fri Apr 25 16:52:43 2008 Subject: Old school 2D gel electrophoresis Message-ID: <481239AF.8050204@bio.umass.edu> I am interested in doing some proteomic work using 2D gel electrophoresis. We have the hardware to run 2D gels, however our equipment is many decades old and uses tube gels to resolve in the first (IEF) dimension. Most of the published in the last 10+ years describes a first dimension separation by immobilized pH strips, which is presumably better. Does anyone in this group have experience using tube gels? Does the newer method truly warrant the costs associated with it (BioRad cost for the power pack is ~$8,000)? Does anyone have any advice to offer in using tube gels before I dive in? Thanks, Arne -- __ Arne K Christensen Postdoctoral Research Associate University of Massachusetts, Amherst USGS Conte Anadromous Fish Research Center One Migratory Way, PO Box 796 Turners Falls, MA 01376 Email: arnec@bio.umass.edu Phone: (413) 863-3827 Fax: (413) 863-9810 URL: www.biobog.com From Nikola.Wenta from nottingham.ac.uk Sat Apr 26 12:25:19 2008 From: Nikola.Wenta from nottingham.ac.uk (Wenta Nikola) Date: Sat Apr 26 15:34:06 2008 Subject: Old school 2D gel electrophoresis (Arne K Christensen) In-Reply-To: <200804261704.m3QH4pT15446@net.bio.net> References: <200804261704.m3QH4pT15446@net.bio.net> Message-ID: Hi Arne, You could at first run a "traditional" 1D-Gel (IEF, in example), then you cut out the lanes and place them on top of a stacking gel (with separating gel below) and run it as a "multilane" sample; to be sure that each lane run properly in the 1st dimension, you could load your sample between two marker lanes. Biorad offers "two lane combs" (165-3356 Mini-PROTEAN Comb, prep/2-D well, 0.75 mm) with one pocket for the marker and one broad "lane" for the gel slice (horizontal). The remaining space around the cut slice, you can fill up with sample buffer. 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 yansun2005 from gmail.com Sat Apr 26 18:38:34 2008 From: yansun2005 from gmail.com (yan sun) Date: Sat Apr 26 19:21:27 2008 Subject: Page gel Message-ID: Dear all, I got a question about PAGE electrophoresis gel to confirm. When we mention that proper volts condition for running a gel is around 20V/cm. For eample, my gel is 20 cm, which means that proper Volts is ~400V? Am I right? If the volts is toooo high, the gel might toooo hot to get good resolution, right? Thx a lot!!!! Frances -- Y. F.S. From David.L.Haviland from uth.tmc.edu Sun Apr 27 10:56:49 2008 From: David.L.Haviland from uth.tmc.edu (Haviland, David L) Date: Sun Apr 27 17:21:55 2008 Subject: Page gel References: Message-ID: <76E50A283589324FA6A1999EEFBB1341011A181A@UTHEVS1.mail.uthouston.edu> The reasons you cite is exactly why I always ran my gels on constant amps rather than volts. For a 10% SDS-PAGE I could start it at 5 pm and by 9 am the next morning it was about 3 cm from the bottom. Even during the day I never ran them above 25 mA without some form of cooling else they'd "smile" and make interpretation a bit problematic. In my hands, slower was always better. David -----Original Message----- From: methods-bounces@oat.bio.indiana.edu on behalf of yan sun Sent: Sat 4/26/2008 6:38 PM To: Methods@magpie.bio.indiana.edu Subject: Page gel Dear all, I got a question about PAGE electrophoresis gel to confirm. When we mention that proper volts condition for running a gel is around 20V/cm. For eample, my gel is 20 cm, which means that proper Volts is ~400V? Am I right? If the volts is toooo high, the gel might toooo hot to get good resolution, right? Thx a lot!!!! Frances -- Y. F.S. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From Nikola.Wenta from nottingham.ac.uk Sun Apr 27 12:14:12 2008 From: Nikola.Wenta from nottingham.ac.uk (Wenta Nikola) Date: Sun Apr 27 17:22:03 2008 Subject: Page gel In-Reply-To: <200804271703.m3RH3sQ12520@net.bio.net> References: <200804271703.m3RH3sQ12520@net.bio.net> Message-ID: Hi Yan, you are right; I think you are talking about EMSA or other native gel systems that use larger gels instead of the Biorad mini gels, right? We run out gels also at 400 V, but in the cold room. Another possibility would be water cooling as realized with the Protean ii XL. On the other hand, you can run that gel at 400 V, but as you mentioned, it would become hot; therefore, I wouldn't be concerned of the resolution but more about breakage of glas. 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 R.Jayakumar from roswellpark.org Sun Apr 27 14:55:33 2008 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Sun Apr 27 17:22:10 2008 Subject: Page gel In-Reply-To: References: Message-ID: <97101976F8A044468CA74FE11883B90E173E7A52@VISTA.roswellpark.org> That is why we have cooooooling circulating water baths to run our laaaaaaaaaarge SDS PAGE gels. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of yan sun Sent: Saturday, April 26, 2008 7:39 PM To: Methods@magpie.bio.indiana.edu Subject: Page gel Dear all, I got a question about PAGE electrophoresis gel to confirm. When we mention that proper volts condition for running a gel is around 20V/cm. For eample, my gel is 20 cm, which means that proper Volts is ~400V? Am I right? If the volts is toooo high, the gel might toooo hot to get good resolution, right? Thx a lot!!!! Frances -- Y. F.S. _______________________________________________ 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 yansun2005 from gmail.com Sun Apr 27 22:39:11 2008 From: yansun2005 from gmail.com (yan sun) Date: Tue Apr 29 11:32:23 2008 Subject: Page gel In-Reply-To: <76E50A283589324FA6A1999EEFBB1341011A181A@UTHEVS1.mail.uthouston.edu> References: <76E50A283589324FA6A1999EEFBB1341011A181A@UTHEVS1.mail.uthouston.edu> Message-ID: run 20% gel 4w around 10 hours, but the ladder is diffused, I mean the band is really wide"( did it happen to you? if you run under very low watage, it is easy to diffuse. On 4/27/08, Haviland, David L wrote: > > > The reasons you cite is exactly why I always ran my gels on constant amps > rather than volts. For a 10% SDS-PAGE I could start it at 5 pm and by 9 am > the next morning it was about 3 cm from the bottom. Even during the day I > never ran them above 25 mA without some form of cooling else they'd "smile" > and make interpretation a bit problematic. In my hands, slower was always > better. > > David > > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu on behalf of yan sun > Sent: Sat 4/26/2008 6:38 PM > To: Methods@magpie.bio.indiana.edu > Subject: Page gel > > Dear all, > I got a question about PAGE electrophoresis gel to confirm. > When we mention that proper volts condition for running a gel is around > 20V/cm. > For eample, my gel is 20 cm, which means that proper Volts is ~400V? Am I > right? > If the volts is toooo high, the gel might toooo hot to get good > resolution, > right? > Thx a lot!!!! > > Frances > -- > Y. F.S. > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > -- Y. F.S. From itisam.sarangi from gmail.com Mon Apr 28 12:05:24 2008 From: itisam.sarangi from gmail.com (Itisam Sarangi) Date: Tue Apr 29 11:32:35 2008 Subject: Methods Digest, Vol 35, Issue 4 In-Reply-To: <200804081704.m38H4aT19020@net.bio.net> References: <200804081704.m38H4aT19020@net.bio.net> Message-ID: <25de2fb70804281005l45a179bcj44a38dac0d4852b9@mail.gmail.com> Dear all I am trying to purifying a DNA binding protein from E.coli..and trying to remove the contaminating DNA... I am using DNAse to remove the contaminating DNA ....but the protein is precipitaing in the contact with diavalent cations used for DNAse activity...if any one have suggestions plz feel free to share.... itisam From itisam.sarangi from gmail.com Mon Apr 28 12:13:37 2008 From: itisam.sarangi from gmail.com (Itisam Sarangi) Date: Tue Apr 29 11:32:41 2008 Subject: DNA binding protein purification Message-ID: <25de2fb70804281013n4a990de1t3f896516380e386@mail.gmail.com> Dear all I am a regular reader of the methods list. presently I am trying to purifying a DNA binding protein from E.coli..and trying to remove the contaminating DNA... I am using DNAse to remove the contaminating DNA ....but the protein is precipitaing in the contact with diavalent cations used for DNAse activity...if any one have suggestions plz feel free to share.... itisam From engelbert_buxbaum from hotmail.com Mon Apr 28 14:17:02 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Apr 29 11:32:46 2008 Subject: Old school 2D gel electrophoresis References: Message-ID: Am 25.04.2008, 16:06 Uhr, schrieb Arne K Christensen : > I am interested in doing some proteomic work using 2D gel > electrophoresis. We have the hardware to run 2D gels, however our > equipment is many decades old and uses tube gels to resolve in the first > (IEF) dimension. Most of the published in the last 10+ years describes > a first dimension separation by immobilized pH strips, which is > presumably better. Yes, and for good reasons: - the flat gels use immobilised pH-gradients, which do not show cathodic drift and work for protein with more alkaline pI. They are also more reproducible than the ampholine based ones. - cooling is much more effective - in traditional tube gels the protein is added at the acidic side. In my experience a lot of protein precipitates under these conditions and doesn't enter the gel at all. In flat gels you can add the sample at whatever pH you choose (or simply soak the sample into the gel during the rehydration phase) - tube gels tend to break when you push them out of the tube (or do not come out in the first place). Flat gels have a plastic backing, which is almost indestructible. > Does anyone in this group have experience using tube gels? Does the > newer method truly warrant the costs associated with it (BioRad cost for > the power pack is ~$8,000)? IMHO unequivocally yes, if you have to do it more than 2 or three times. But if you want to fiddle with the tubes (just to see how heroic those times were), at least polymerise a cotton thread into the gel for easier handling (Patton et al., Biotechniques 8 (1990) 518-27). Btw, I was a very satisfied customer of the Pharmacia IGPhor unit, no idea whether it's still on the market (Pharmacia was bought by GE). From Nikola.Wenta from nottingham.ac.uk Tue Apr 29 12:36:55 2008 From: Nikola.Wenta from nottingham.ac.uk (Wenta Nikola) Date: Tue Apr 29 17:55:35 2008 Subject: DNA binding protein purification References: <200804291704.m3TH4MQ26052@net.bio.net> Message-ID: Hello Itisam, our lab also works with DNA binding proteins. We isolate that protein from Sf9 cells and also from bacteria cells. After lysis, we don't do DNase digest but instead use 0.1% (v/v) PEE (Polyethylenimine) from Sigma (P-3143) to get rid of DNA. For our protein, it works well; maybe you need to check your protein's pI because PEE might also precipitate your protein ;-). 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 R.Jayakumar from roswellpark.org Tue Apr 29 12:44:13 2008 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Tue Apr 29 17:55:43 2008 Subject: Page gel In-Reply-To: References: <76E50A283589324FA6A1999EEFBB1341011A181A@UTHEVS1.mail.uthouston.edu> Message-ID: <97101976F8A044468CA74FE11883B90E173E7A55@VISTA.roswellpark.org> I think he meant VOLTS or Amp and not WATTS. If you run it at 4 Watts, that must be a very low volts. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of yan sun Sent: Sunday, April 27, 2008 11:39 PM To: Haviland, David L Cc: Methods@magpie.bio.indiana.edu Subject: Re: Page gel run 20% gel 4w around 10 hours, but the ladder is diffused, I mean the band is really wide"( did it happen to you? if you run under very low watage, it is easy to diffuse. On 4/27/08, Haviland, David L wrote: > > > The reasons you cite is exactly why I always ran my gels on constant amps > rather than volts. For a 10% SDS-PAGE I could start it at 5 pm and by 9 am > the next morning it was about 3 cm from the bottom. Even during the day I > never ran them above 25 mA without some form of cooling else they'd "smile" > and make interpretation a bit problematic. In my hands, slower was always > better. > > David > > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu on behalf of yan sun > Sent: Sat 4/26/2008 6:38 PM > To: Methods@magpie.bio.indiana.edu > Subject: Page gel > > Dear all, > I got a question about PAGE electrophoresis gel to confirm. > When we mention that proper volts condition for running a gel is > around 20V/cm. > For eample, my gel is 20 cm, which means that proper Volts is ~400V? > Am I right? > If the volts is toooo high, the gel might toooo hot to get good > resolution, right? > Thx a lot!!!! > > Frances > -- > Y. F.S. > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > -- Y. F.S. _______________________________________________ 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 kgilbride from shire.com Wed Apr 30 09:15:52 2008 From: kgilbride from shire.com (Gilbride, Kevin) Date: Wed Apr 30 12:50:01 2008 Subject: Page gel In-Reply-To: Message-ID: <0F2CFCE6FC067A42813DFA1F9BB0697C01099876@SHCHBEX03.corp.shire.com> 20% is high for acrylamide. Have you considered a tricine gel? If you absolutely need sds page 4c @ 18mA overnight should work fine. Also, if you are using a premixed concentrate for your running buffer the pH can be affected by your water source. K -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of yan sun Sent: Sunday, April 27, 2008 11:39 PM To: Haviland, David L Cc: Methods@magpie.bio.indiana.edu Subject: Re: Page gel run 20% gel 4w around 10 hours, but the ladder is diffused, I mean the band is really wide"( did it happen to you? if you run under very low watage, it is easy to diffuse. On 4/27/08, Haviland, David L wrote: > > > The reasons you cite is exactly why I always ran my gels on constant amps > rather than volts. For a 10% SDS-PAGE I could start it at 5 pm and by 9 am > the next morning it was about 3 cm from the bottom. Even during the day I > never ran them above 25 mA without some form of cooling else they'd "smile" > and make interpretation a bit problematic. In my hands, slower was always > better. > > David > > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu on behalf of yan sun > Sent: Sat 4/26/2008 6:38 PM > To: Methods@magpie.bio.indiana.edu > Subject: Page gel > > Dear all, > I got a question about PAGE electrophoresis gel to confirm. > When we mention that proper volts condition for running a gel is around > 20V/cm. > For eample, my gel is 20 cm, which means that proper Volts is ~400V? Am I > right? > If the volts is toooo high, the gel might toooo hot to get good > resolution, > right? > Thx a lot!!!! > > Frances > -- > Y. F.S. > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > -- Y. F.S. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods ______________________________________________________________________ This email has been scanned by the MessageLabs Email Security System. For more information please visit http://www.messagelabs.com/email ______________________________________________________________________ Please consider the environment before printing this e-mail This email and any files transmitted with it are confidential and may be legally privileged and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient please note that any disclosure, distribution, or copying of this email is strictly prohibited and may be unlawful. If received in error, please delete this email and any attachments and confirm this to the sender. www.shire.com From joseolucha from gmail.com Wed Apr 30 22:27:52 2008 From: joseolucha from gmail.com (Jose Olucha) Date: Thu May 1 10:33:50 2008 Subject: Absorbance/ Fluorescence for detection of N-formyl-hydroxy amino acids Message-ID: Dear all, Does anyone know of a good method for detecting (I'm thinking absorbance / fluorescence) N-formyl-hydroxyamino acids (eg. N-formyl-hydroxy-lysine)? If not, a removal of the formyl group that doesn't involve boiling highly concentrated acids? Single amino acid deformylases? Have searched literature to no avail.... A positive answer would make a grad student's life so much better!! Regards, Jose