From L.Kamalian from liverpool.ac.uk Mon Jun 2 03:51:50 2008 From: L.Kamalian from liverpool.ac.uk (Kamalian, Laleh) Date: Mon Jun 2 11:00:06 2008 Subject: Proliferation assay in suspension cells References: <31DC2DFFD70C174B8E3072803F894F91046C8C@EVSSTAFF1.livad.liv.ac.uk> <2A67EA781EC7F949A2AB0A0D07A86C6A029729D2@mail01.ccia.local> Message-ID: <31DC2DFFD70C174B8E3072803F894F91046C8E@EVSSTAFF1.livad.liv.ac.uk> Dear Scott Thanks for your reply. How do you use MTT in suspension cells? My underestanding is that I would have to spin down the cells each day to be able to add MTT. Wouldn't that cause loosing some of the cells and making the test unreliable, especially in the case of wells with very low number of cells (<1000) in which cell pellets would be hardly visible after spinning? Thanks again. Laleh ________________________________ From: Scott Brown [mailto:SBrown@ccia.unsw.edu.au] Sent: Sat 31/05/2008 02:37 To: Kamalian, Laleh Subject: RE: Proliferation assay in suspension cells MTT can be used for suspension cells, i use Nalm6 cells regularly for MTT assays, i find the 96 well plates with the more concave well works better. Scott ________________________________ From: methods-bounces@oat.bio.indiana.edu on behalf of Kamalian, Laleh Sent: Fri 5/30/2008 9:21 PM To: methods Subject: Proliferation assay in suspension cells Hi and thanks to everyone who have been helping me so far. I am in trouble again. I need a protocol for proliferation assay in cells that grow in suspension. The method that is used in our lab is MTT assay which is as I underestand for adherent cells. Thanks Laleh _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From prae from gmx.net Tue Jun 3 04:50:55 2008 From: prae from gmx.net (Christian Praetorius) Date: Mon Jun 2 11:00:12 2008 Subject: Polaroid 667 instant films replacement? References: Message-ID: <6ahu4bF36fdthU1@mid.individual.net> "AK" wrote: > Fujifilm FP-3000B Professional Instant Black & White Film ISO >3000 (3.25 x 4.25", Glossy) Single Pack (10 exp.) > > Mfr# 15200772 . B&H# FUFP3000B >you may check with them if it will fit your camera. unfortunately its out of >stock, but hope not out of business. >good luck This should be a good replacement. And as far as I know, Fuji does not plan to discontinue the film. The other possibility would be to buy a number of films and put them in stock. They can be stored for quite a long time (I used films which ran out ten years ago), but there is no guarantee that this will work. And: Never freeze this films, only put them into a fridge. Christian -- X-no-Sig: yes From pow.joshi from gmail.com Mon Jun 2 17:29:20 2008 From: pow.joshi from gmail.com (Pow Joshi) Date: Mon Jun 2 17:52:18 2008 Subject: RNA Later-Crystal!! In-Reply-To: References: Message-ID: <710764ea0806021529x59c32b5fi7a3747d181a6edc7@mail.gmail.com> 2008/5/30 Sudheendra Rao N R : > Hi all, > I had kept RNA later at room temperature in an open petri dish by mistake > and i saw almost colorless longitudinal (not smooth but did not have any > defined number of surfaces also) crystals.. of varying length..not soft but > easily breakable > what are they? I tried heating the crystal to 50 degrees for 10 min but it > did not melt..but when i heated to 100 degrees it melted and > evaporatorated. > i guess it must be a molecular crystal! > can u guys shed more light on it? woooooooooooow .... that's some magic :)) I wonder if it's just salt or sds or whatever else that may be in the RNA sln..... or perhaps you're wondering if the RNA crystalised? Pow > > > Sudheendra. > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From engelbert_buxbaum from hotmail.com Tue Jun 3 07:50:57 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Jun 3 12:27:11 2008 Subject: Questions about protein quantification - from the industry perspective References: Message-ID: Am 27.05.2008, 07:48 Uhr, schrieb Aawara Chowdhury : > In , > Trond Erik Vee Aune wrote: > How about using beta-lactamase? Here's the original reference from > Roger Tsien's group - chloramphenicol O-acetyltransferase (CAT) would also come to mind, but has a molecular mass of about 70 kDa From engelbert_buxbaum from hotmail.com Tue Jun 3 08:19:46 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Jun 3 12:27:16 2008 Subject: can i use EDTA instead of TEMED for SDS PAGE ? References: <6I5%j.233$k67.12@newsfe02.lga> Message-ID: Am 30.05.2008, 20:50 Uhr, schrieb DK : > Yeah, it's one of the biggest ironies of science techniques. The actual > buffer system is what is properly referred to as Ornstein-Davis > system, developed some time in 1960s. Leonard Ornstein, who also > shares a credit for developing acrylamide gels to begin with (!), used > to occasionally post here a while ago. All Laemmli did was to add SDS > to this buffer system - with, as we know, quite spectacular results. > > All the pertinent details and refs can be found by digging at Ornstein's > web page: http://www.pipeline.com/~lenornst/ > > Sic transit gloria mundi. Hardly anyone these days knows his name. > Then again, it's not like Tiselius is a household name either. Well, the Ornstein & Davis paper introduced DISK electrophoresis (based on work by several other authors, going back to Kohlrausch's 1897 paper on what now we call isotachophoresis). The concentrating effect of DISK-electrophoresis - or rather the narrow bands resulting from it - are only one reason for the success of the Laemli method. The other is that since SDS (and some other detergents like CTAB) bind to protein in an approximately constant ratio of 1 detergent to 3 amino acids. Thus all proteins have almost the same charge to weight ratio and experience the same accellerating force in a field. Also, because of the denaturing effect of SDS and BME all proteins have roughly the same shape. So in Laemmli's method of the 3 factors that determine electrophoretic mobility (size, shape and charge) only size remains. This makes the gels interpretable, and that constitutes considerable progress. By the way, although in Laemmli's 1970 Nature paper the discussion of the method is indeed rather brief and limited to a figure legend, Laemmli & Favre (J. Mol. Biol. 80 (1973) 575-599) gives more info and I have argued that it is that paper that should be cited for the method. From engelbert_buxbaum from hotmail.com Tue Jun 3 10:01:37 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Jun 3 12:27:20 2008 Subject: can i use EDTA instead of TEMED for SDS PAGE ? References: <6I5%j.233$k67.12@newsfe02.lga> <3915730b-7951-4afd-bc8f-bb5a8b23e543@y21g2000hsf.googlegroups.com> Message-ID: Am 30.05.2008, 16:35 Uhr, schrieb WS : > Dear Hiranya, > > is there any chance to obtain this paper? I am (sort of desperately) > looking for literature which really explains what is the chemistry and > physics behind the different gel systems (Tris-Glycine, Tris-Tricine, > how stacking gels work, what improves and what worsens the resolution > etc.). > > Are there any other systems that add copolymers to acrylamide or use > something completely different? (You can see, I really want to mess it > up...) For the theory, look at the following papers: @article{Jov-73a, AUTHOR= {T.M. Jovin}, TITLE= {Multiphasic Zone Electrophoresis. {I}. {S}teady-State Moving-Boundary Systems Formed by Different Electrolyte Combinations}, JOURNAL= {Biochemistry}, VOLUME= {12}, YEAR= {1973}, PAGES= {871-879}, LANGUAGE= {engl} } @article{Jov-73b, AUTHOR= {T.M. Jovin}, TITLE= {Multiphasic Zone Electrophoresis. {II}. Design of Integrated Discontinuous Buffer Systems for Analytical and Preparative Fractionation}, JOURNAL= {Biochemistry}, VOLUME= {12}, YEAR= {1973}, PAGES= {879-890}, LANGUAGE= {engl} } @article{Jov-73c, AUTHOR= {T.M. Jovin}, TITLE= {Multiphasic Zone Electrophoresis. {III}. {F}urther Analysis and New Forms of Discontinuous Buffer Systems}, JOURNAL= {Biochemistry}, VOLUME= {12}, YEAR= {1973}, PAGES= {890-898}, LANGUAGE= {engl} } @article{Jov-73d, AUTHOR= {T.M. Jovin}, TITLE= {Multiphasic zone electrophoresis. {IV} {D}esign and analysis of discontinuous buffer systems with a digital computer}, JOURNAL= {Ann. N.Y. Acad. Sci.}, VOLUME= {209}, YEAR= {1973}, PAGES= {477-496}, LANGUAGE= {engl} } There is a web site for the calculation of buffer systems satisfying certain criteria at http://www.buffers.nichd.nih.gov/, but it currently seems non-operational. You can of cdourse use different detergents, in particular those with positive charge. As I explained in the following paper they have advantages with certain types of proteins: @article{Bux-03, AUTHOR= {E. Buxbaum}, TITLE= {Cationic Electrophoresis and Electrotransfer of Membrane Glycoproteins}, YEAR= {2003}, JOURNAL= {Anal. Biochem.}, PAGES= {70-76}, VOLUME= {314}, LANGUAGE= {engl}, DOI= {10.1016/S0003-2697(02)00639-5} } In theory one could also link affinity tags to the matrix to get an affinity electrophoresis in analogy of affinity chromatography, but I am not aware of anybody ever having done that. Other than that the matrix only has to provide the right pore size, its chemical composition is not relevant. So starch or agar gels are used for some problems, but only because they offer larger pores than acrylamide. From engelbert_buxbaum from hotmail.com Tue Jun 3 10:09:33 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Jun 3 12:27:26 2008 Subject: Proliferation assay in suspension cells References: Message-ID: Am 30.05.2008, 07:21 Uhr, schrieb Kamalian, Laleh : > Hi and thanks to everyone who have been helping me so far. I am in > trouble again. I need a protocol for proliferation assay in cells that > grow in suspension. The method that is used in our lab is MTT assay > which is as I underestand for adherent cells. You can also use it for suspension cells, the only problem is that you have to replace the medium by the MTT solution and the MTT solution by the propanol/HCl reagent. That is a lot easier with anchored cells where you simply flick of the solution to be discarded. With suspended cells you'll have to spin, which is more work. But any other assay you might use suffers from the same problem. From sudhee26 from gmail.com Mon Jun 2 22:56:18 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Tue Jun 3 12:30:35 2008 Subject: RNA Later-Crystal!! In-Reply-To: <710764ea0806021529x59c32b5fi7a3747d181a6edc7@mail.gmail.com> References: <710764ea0806021529x59c32b5fi7a3747d181a6edc7@mail.gmail.com> Message-ID: Well, I asked ambion guys. they said that RNAlater being super-saturated salt solution, crystallization is possible if there is any evoparation. That settles the stuff :) somewhere i read that RNAlater contains ammonium sulphate well..it must be something else..i checked out ammonium sulphate crystals..they are not long..yup but colorless.. On Tue, Jun 3, 2008 at 3:59 AM, Pow Joshi wrote: > > > 2008/5/30 Sudheendra Rao N R : > >> Hi all, >> I had kept RNA later at room temperature in an open petri dish by mistake >> and i saw almost colorless longitudinal (not smooth but did not have any >> defined number of surfaces also) crystals.. of varying length..not soft >> but >> easily breakable >> what are they? I tried heating the crystal to 50 degrees for 10 min but it >> did not melt..but when i heated to 100 degrees it melted and >> evaporatorated. >> i guess it must be a molecular crystal! >> can u guys shed more light on it? > > > woooooooooooow .... that's some magic :)) I wonder if it's just salt or sds > or whatever else that may be in the RNA sln..... or perhaps you're wondering > if the RNA crystalised? > > Pow > >> >> >> Sudheendra. >> >> -- >> Think before agree >> Think before you nod >> but STOP thinking >> and You Are God >> _______________________________________________ >> Methods mailing list >> Methods@net.bio.net >> http://www.bio.net/biomail/listinfo/methods >> > > -- Think before agree Think before you nod but STOP thinking and You Are God From sioklian from gmail.com Tue Jun 3 00:13:49 2008 From: sioklian from gmail.com (lsl) Date: Tue Jun 3 12:30:39 2008 Subject: Spot Density on DNA Microarray Message-ID: <6384f179-7427-496b-a297-7653434103ae@w8g2000prd.googlegroups.com> Dear all, Do you have any idea for the commercially available DNA microarray, what is the oligo density of each of the spot (i.e. molecules/spot)? Appreciate if you can give me some literatures which mention about that. Thank you very much. From trondaun from REMOVE.nt.ntnu.no Tue Jun 3 01:54:49 2008 From: trondaun from REMOVE.nt.ntnu.no (Trond Erik Vee Aune) Date: Tue Jun 3 12:30:44 2008 Subject: Questions about protein quantification - from the industry perspective In-Reply-To: <94015b8a-c9b1-494a-af7a-523bf09fb045@a70g2000hsh.googlegroups.com> References: <94015b8a-c9b1-494a-af7a-523bf09fb045@a70g2000hsh.googlegroups.com> Message-ID: WS skrev: > Hi Trond, > > what about beta-galactosidase or beta-glucuronidase? There are > chromogenic and fluorogenic subtrates available. Are you suggesting using the alpha subunit of beta-galactosidase as a reporter tag (analogous to blue/white screening)? If so I worry that the signal strength will be inhibited by low levels of the omega subunit (expressed from the chromosome). And I don't know if this will function at all in other bacteria except E. coli. Another possibility is of course to use the whole beta-galactosidase as a reporter, and this is presumably what you meant, but unfortunately this protein is very large (1021 aa) and might therefore not be suitable as a reporter tag (?). And would the presence of chromosomal copy raise the background too high? This last question is relevant for beta-glucuronidase too. Unfortunately, in addition, use of the GUS assay is covered by patents, so this might be an expensive alternative. Trond Erik From trondaun from REMOVE.nt.ntnu.no Tue Jun 3 01:55:07 2008 From: trondaun from REMOVE.nt.ntnu.no (Trond Erik Vee Aune) Date: Tue Jun 3 12:30:49 2008 Subject: Questions about protein quantification In-Reply-To: References: Message-ID: Beug, Shawn skrev: > > -----Original Message----- > Why not use alkaline phosphatase? It works very well in my hands. A > commercial source is www.genhunter.com It seems to be this Genhunter technology is developed for detection of ligands and receptors, do you know if it functions on intracellular proteins? Trond Erik From blackhole from abuse.plus.com Tue Jun 3 11:27:30 2008 From: blackhole from abuse.plus.com (Duncan Clark) Date: Tue Jun 3 12:30:59 2008 Subject: RNA Later-Crystal!! References: Message-ID: Historians believe that in newspost on Mon, 2 Jun 2008, Pow Joshi penned the following literary masterpiece: >2008/5/30 Sudheendra Rao N R : > >> Hi all, >> I had kept RNA later at room temperature in an open petri dish by mistake >> and i saw almost colorless longitudinal (not smooth but did not have any >> defined number of surfaces also) crystals.. of varying length..not soft but >> easily breakable >> what are they? I tried heating the crystal to 50 degrees for 10 min but it >> did not melt..but when i heated to 100 degrees it melted and >> evaporatorated. >> i guess it must be a molecular crystal! >> can u guys shed more light on it? > > >woooooooooooow .... that's some magic :)) I wonder if it's just salt or sds >or whatever else that may be in the RNA sln..... or perhaps you're wondering >if the RNA crystalised? RNALater is AmmSO4 based isn't it? Ambion patent/patent app. should have exact details. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From ebs15242 from creighton.edu Tue Jun 3 10:13:23 2008 From: ebs15242 from creighton.edu (Ed Siefker) Date: Tue Jun 3 12:31:05 2008 Subject: RNA Later-Crystal!! In-Reply-To: References: Message-ID: <48455F93.2070400@creighton.edu> You can find the composition of RNA later at : http://www.protocol-online.org/biology-forums/posts/8295.html It's something like 5 molar (NH4)2SO4, so I'd bet that's what your crystal is. Note: I haven't tried the recipe for homemade RNALater. I'd like to hear from anyone who does though. Sudheendra Rao N R wrote: > Hi all, > I had kept RNA later at room temperature in an open petri dish by mistake > and i saw almost colorless longitudinal (not smooth but did not have any > defined number of surfaces also) crystals.. of varying length..not soft but > easily breakable > what are they? I tried heating the crystal to 50 degrees for 10 min but it > did not melt..but when i heated to 100 degrees it melted and evaporatorated. > i guess it must be a molecular crystal! > can u guys shed more light on it? > > Sudheendra. > From sbeug from ohri.ca Tue Jun 3 14:36:33 2008 From: sbeug from ohri.ca (Beug, Shawn) Date: Tue Jun 3 14:53:17 2008 Subject: Questions about protein quantification (Trond Erik Vee Aune) In-Reply-To: <200806031731.m53HVUO19645@net.bio.net> Message-ID: <7C08583D5DA01D4D963926046238C6C7082636@ohexch05.civic1.ottawahospital.on.ca> It all depends on what you are trying to detect. It is possible to detect soluble ligand in the secretory pathway through this method (which is partly why I am using this system). The best references I have found to date are: Methods in Enzymology 327:19-35 and 327:198-210 Sci. STKE. 168:p12 Cheers, Shawn > -----Original Message----- > Why not use alkaline phosphatase? It works very well in my hands. A > commercial source is www.genhunter.com It seems to be this Genhunter technology is developed for detection of ligands and receptors, do you know if it functions on intracellular proteins? Trond Erik -------------------- Confidentiality Statement - The contents of this e-mail, including its attachment, are intended for the exclusive use of the recipient and may contain confidential or privileged information. If you are not the intended recipient, you are strictly prohibited from reading, using, disclosing, copying, or distributing this e-mail or any of its contents. If you received this e-mail in error, please notify the sender by reply e-mail immediately or the Privacy Office (privacy@ottawahospital.on.ca ) and permanently delete this e-mail and its attachments, along with any copies thereof. Thank you. Avis de confidentialité – Ce courriel, y compris ses pièces jointes, s’adresse au destinataire uniquement et pourrait contenir des renseignements confidentiels. Si vous n’êtes pas le bon destinataire, il est strictement interdit de lire, d’utiliser, de divulguer, de copier ou de diffuser ce courriel ou son contenu, en partie ou en entier. Si vous avez reçu ce courriel par erreur, veuillez en informer immédiatement l’expéditeur ou le bureau de la Protection des renseignements personnels (info.privee@hopitalottawa.on.ca), puis effacez le courriel ainsi que les pièces jointes et toute autre copie. Merci. -------------------- From rory.obrien from stonebow.otago.ac.nz Tue Jun 3 20:35:31 2008 From: rory.obrien from stonebow.otago.ac.nz (Rory O'Brien) Date: Wed Jun 4 10:07:19 2008 Subject: RNA Later-Crystal!! Message-ID: I did try making it up according to http://www.protocol-online.org/biology-forums/posts/8295.html but there was just no way that amount of Ammonium sulphate would dissolve... I also came across this http://www.nature.com/labinvest/journal/v83/n1/full/3780599a.html Looks like plain ol' PBS is just as effective ;-) No, I haven't tried it, yet... - Rory. > >> Hi all, > >> I had kept RNA later at room temperature in an open petri dish by > >> mistake and i saw almost colorless longitudinal (not > smooth but did > >> not have any defined number of surfaces also) crystals.. > of varying > >> length..not soft but easily breakable what are they? I > tried heating > >> the crystal to 50 degrees for 10 min but it did not > melt..but when i > >> heated to 100 degrees it melted and evaporatorated. > >> i guess it must be a molecular crystal! > >> can u guys shed more light on it? > > > > > >woooooooooooow .... that's some magic :)) I wonder if it's > just salt or > >sds or whatever else that may be in the RNA sln..... or > perhaps you're > >wondering if the RNA crystalised? > > RNALater is AmmSO4 based isn't it? Ambion patent/patent app. > should have exact details. > > Duncan > -- > I love deadlines. I especially like the whooshing noise they make as > they go flying by. > > Duncan Clark > GeneSys Ltd. > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods From rory.obrien from stonebow.otago.ac.nz Tue Jun 3 23:59:36 2008 From: rory.obrien from stonebow.otago.ac.nz (Rory O'Brien) Date: Wed Jun 4 10:07:33 2008 Subject: RNA Later-Crystal!! Message-ID: I did try making it up according to http://www.protocol-online.org/biology-forums/posts/8295.html but there was just no way that amount of Ammonium sulphate would dissolve... I also came across this http://www.nature.com/labinvest/journal/v83/n1/full/3780599a.html Looks like plain ol' PBS is just as effective ;-) No, I haven't tried it, yet... - Rory. > >> Hi all, > >> I had kept RNA later at room temperature in an open petri dish by > >> mistake and i saw almost colorless longitudinal (not > smooth but did > >> not have any defined number of surfaces also) crystals.. > of varying > >> length..not soft but easily breakable what are they? I > tried heating > >> the crystal to 50 degrees for 10 min but it did not > melt..but when i > >> heated to 100 degrees it melted and evaporatorated. > >> i guess it must be a molecular crystal! > >> can u guys shed more light on it? > > > > > >woooooooooooow .... that's some magic :)) I wonder if it's > just salt or > >sds or whatever else that may be in the RNA sln..... or > perhaps you're > >wondering if the RNA crystalised? > > RNALater is AmmSO4 based isn't it? Ambion patent/patent app. > should have exact details. > > Duncan From novalidaddress from nurfuerspam.de Wed Jun 4 04:35:49 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Jun 4 10:07:37 2008 Subject: can i use EDTA instead of TEMED for SDS PAGE ? References: <6I5%j.233$k67.12@newsfe02.lga> <3915730b-7951-4afd-bc8f-bb5a8b23e543@y21g2000hsf.googlegroups.com> Message-ID: <9563d0b5-bb82-4ee5-99a1-0b486171583f@i76g2000hsf.googlegroups.com> 100 points, Engelbert! The papers are even available online: http://linkinghub.elsevier.com/retrieve/pii/S0003269702006395 http://pubs.acs.org/cgi-bin/archive.cgi/bichaw/1973/12/i05/pdf/bi00729a016.pdf http://pubs.acs.org/cgi-bin/archive.cgi/bichaw/1973/12/i05/pdf/bi00729a015.pdf http://pubs.acs.org/cgi-bin/archive.cgi/bichaw/1973/12/i05/pdf/bi00729a014.pdf http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1749-6632.1973.tb47551.x and some more maybe interesting stuff: http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1164673&blobtype=pdf Best regards, Wolfgang From hroychow from nmsu.edu Wed Jun 4 09:13:14 2008 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Wed Jun 4 10:07:43 2008 Subject: can i use EDTA instead of TEMED for SDS PAGE ? In-Reply-To: References: <6I5%j.233$k67.12@newsfe02.lga> <3915730b-7951-4afd-bc8f-bb5a8b23e543@y21g2000hsf.googlegroups.com> Message-ID: <3630.70.177.43.154.1212588794.squirrel@webmail.nmsu.edu> Thanks for rsponding to this email, Engelbert. I am out of town at the moment and had not had a chance to check my emails regularly. Hiranya > Am 30.05.2008, 16:35 Uhr, schrieb WS : > >> Dear Hiranya, >> >> is there any chance to obtain this paper? I am (sort of desperately) >> looking for literature which really explains what is the chemistry and >> physics behind the different gel systems (Tris-Glycine, Tris-Tricine, >> how stacking gels work, what improves and what worsens the resolution >> etc.). >> >> Are there any other systems that add copolymers to acrylamide or use >> something completely different? (You can see, I really want to mess it >> up...) > > For the theory, look at the following papers: > > @article{Jov-73a, > AUTHOR= {T.M. Jovin}, > TITLE= {Multiphasic Zone Electrophoresis. {I}. {S}teady-State > Moving-Boundary Systems Formed by Different Electrolyte Combinations}, > JOURNAL= {Biochemistry}, > VOLUME= {12}, > YEAR= {1973}, > PAGES= {871-879}, > LANGUAGE= {engl} > } > > @article{Jov-73b, > AUTHOR= {T.M. Jovin}, > TITLE= {Multiphasic Zone Electrophoresis. {II}. Design of > Integrated Discontinuous Buffer Systems for Analytical and Preparative > Fractionation}, > JOURNAL= {Biochemistry}, > VOLUME= {12}, > YEAR= {1973}, > PAGES= {879-890}, > LANGUAGE= {engl} > } > > @article{Jov-73c, > AUTHOR= {T.M. Jovin}, > TITLE= {Multiphasic Zone Electrophoresis. {III}. {F}urther > Analysis and New Forms of Discontinuous Buffer Systems}, > JOURNAL= {Biochemistry}, > VOLUME= {12}, > YEAR= {1973}, > PAGES= {890-898}, > LANGUAGE= {engl} > } > > @article{Jov-73d, > AUTHOR= {T.M. Jovin}, > TITLE= {Multiphasic zone electrophoresis. {IV} {D}esign and > analysis of discontinuous buffer systems with a digital computer}, > JOURNAL= {Ann. N.Y. Acad. Sci.}, > VOLUME= {209}, > YEAR= {1973}, > PAGES= {477-496}, > LANGUAGE= {engl} > } > > There is a web site for the calculation of buffer systems satisfying > certain criteria at > http://www.buffers.nichd.nih.gov/, but it currently seems non-operational. > > You can of cdourse use different detergents, in particular those with > positive charge. As I explained in the following paper they have > advantages with certain types of proteins: > > @article{Bux-03, > AUTHOR= {E. Buxbaum}, > TITLE= {Cationic Electrophoresis and Electrotransfer of Membrane > Glycoproteins}, > YEAR= {2003}, > JOURNAL= {Anal. Biochem.}, > PAGES= {70-76}, > VOLUME= {314}, > LANGUAGE= {engl}, > DOI= {10.1016/S0003-2697(02)00639-5} > } > > In theory one could also link affinity tags to the matrix to get an > affinity electrophoresis in analogy of affinity chromatography, but I am > not aware of anybody ever having done that. Other than that the matrix > only has to provide the right pore size, its chemical composition is not > relevant. So starch or agar gels are used for some problems, but only > because they offer larger pores than acrylamide. > _______________________________________________ > 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 novalidaddress from nurfuerspam.de Wed Jun 4 10:27:22 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Jun 4 12:47:13 2008 Subject: RNA Later-Crystal!! References: Message-ID: <6be94225-900c-4478-ae17-1400d2216ef2@m73g2000hsh.googlegroups.com> You might check here: http://www.freepatentsonline.com/6204375.html?query=PN%2F6204375+OR+6204375&stemming=on its one of the patents in Rory's Citation, there was a dead link Enjoy! Wo From novalidaddress from nurfuerspam.de Wed Jun 4 10:28:53 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Jun 4 12:47:18 2008 Subject: RNA Later-Crystal!! References: Message-ID: <3d2b17f1-472d-42c4-a45e-880165c17e1e@z72g2000hsb.googlegroups.com> You might check here: http://www.freepatentsonline.com/6204375.html?query=PN%2F6204375+OR+6204375&stemming=on its one of the patents in Rory's Citation, there was a dead link Enjoy! Wo From novalidaddress from nurfuerspam.de Wed Jun 4 11:14:05 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Jun 4 12:47:22 2008 Subject: RNA Later-Crystal!! References: Message-ID: <345b22c5-8059-4989-8b7c-968924e3edfe@c58g2000hsc.googlegroups.com> Just had a glance at the patent: Genial things usually are simple: The main principle of RNA later seems to be guess what: Ammonium sulfate precipitation! Just precipitate all the bad RNAses, then they won't bother you. Of course, some more goodies are added: eg EDTA 100% saturation is 541.8g/l solution @ 25degC, so 80g/100ml should not be feasible without special tricks. Or does it mean: Add 80g to 100ml. That would make a total volume of about 150 ml, this just might work. Further reading on ammonium sulfate prec. may be found here: http://www.encorbio.com/protocols/AM-SO4.htm Enjoy! Wo From jleonard.iii from gmail.com Wed Jun 4 16:31:39 2008 From: jleonard.iii from gmail.com (John Leonard) Date: Wed Jun 4 16:45:32 2008 Subject: Affinity purification of antibody against MBP-fusion Message-ID: <83d01d5d0806041431k51563103qc15f8c2a53c475d2@mail.gmail.com> I have an antibody against a protein I'm studying, and I'd like to purify it from the serum to see if I can improve its specificity for my protein in Western, EMSA, ChIP, etc. I have a vector for an MBP fusion of my protein (which I know the antibody binds very well), and I was hoping to use that to affinity purify my antibody. Ideally, I would like to express the bacterial fusion, run it over an amylose column, then use the column-bound MBP fusion to affinity purify my antibody. I have seen similar protocols using His- or GST-tagged proteins, but have not found a protocol for MBP fusions. If anyone has a protocol for something like this, or has another idea for how I can purify my antibody based on specific ligand interaction, I'd greatly appreciate it! From rsreedha from mcw.edu Thu Jun 5 09:52:07 2008 From: rsreedha from mcw.edu (Sreedharan, Rajasree) Date: Thu Jun 5 10:51:09 2008 Subject: Cytochrome C western blot Message-ID: Hello, I saw an old post from you regarding cytochrome c western blotting showing a higher band. Did you figure this out? Was it a tetramer? I have the same problem now and so would like to know. Thanks, Raji Sreedharan MD From rob_williams99 from yahoo.com Fri Jun 6 00:32:02 2008 From: rob_williams99 from yahoo.com (Rob Williams) Date: Fri Jun 6 11:45:26 2008 Subject: lambda phage antibody Message-ID: <993884.6843.qm@web53309.mail.re2.yahoo.com> Hi Liselotte, I am also looking for an antibody against Lambda phage. Did you find a company that sells it? Any information you might have would help me greatly. Thank you, Rob Williams rob_williams99@yahoo.com From guojj from missouri.edu Fri Jun 6 12:10:51 2008 From: guojj from missouri.edu (Guo, Jennifer J.) Date: Fri Jun 6 13:01:37 2008 Subject: Cytofluor II (was: "Fluoroscan II software") Message-ID: <62C002D4040DAF4187F1B9B647A8F9E605444AB3@UM-XMAIL03.um.umsystem.edu> Do you still need the Cytofluor II software? From huang_wen from ustc.edu Mon Jun 9 14:45:19 2008 From: huang_wen from ustc.edu (Wen Huang) Date: Mon Jun 9 15:25:12 2008 Subject: Ampicillin Message-ID: <8BDEEB82-30A2-4B48-A871-51FE0FE33F43@ustc.edu> Hi, I was wondering what kind of ampicillin should I use to make LB-amp+ media and LB-agar plates I searched the internet and there are three versions of amplicillins 1. ampicillin 2. ampicillin sodium salt 3. ampicillin trihydrate I am pretty sure people use ampicillin sodium salt for cell culture, just want to make sure that what I should use for bacteria culture. By the way, when these things go into water, do they have the same activity? Thanks, Wen From marina.gouttesoulard from lonza.com Tue Jun 10 08:30:13 2008 From: marina.gouttesoulard from lonza.com (Gouttesoulard Marina - Saint-Beauzire ) Date: Tue Jun 10 09:06:12 2008 Subject: Inactivation of DNase I Message-ID: <2751B10B7FB0A849860E80F52E796B490362A41F@chvex24.lonzagroup.net> Hello, Have you solved your inactivation DNase problem? I have also a problem in inactivation of DNase. I inactive DNase by heating step 10min at 75?C. To check if the inactivation is efficient, I add 1ng DNA in my inactivated sample and I compare the results obtained in PCR with a control containing 1 ng DNA. There is a shift of 2 cycles with the inactivated sample. So I think that the Dnase is not totally inactivated and act on DNA added to destroy it. Have you got some solution to inactivate Dnase without damage RNA. Thanks a lot. rq : the Dnase that I used was the Turbo Dnase from ambion and I already test the inactivator of ambion's kit. Marina > mailto:marina.gouttesoulard@lonza.com > http://www.lonza.com > > This communication and its attachments, if any, may contain confidential and privileged information the use of which by other persons or entities than the intended recipient is prohibited. If you receive this transmission in error, please contact the sender immediately and delete the material from your system. From R.Jayakumar from roswellpark.org Tue Jun 10 09:26:39 2008 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Tue Jun 10 13:32:10 2008 Subject: Ampicillin In-Reply-To: <8BDEEB82-30A2-4B48-A871-51FE0FE33F43@ustc.edu> References: <8BDEEB82-30A2-4B48-A871-51FE0FE33F43@ustc.edu> Message-ID: <97101976F8A044468CA74FE11883B90E173E7A80@VISTA.roswellpark.org> The sodium salt. -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Wen Huang Sent: Monday, June 09, 2008 3:45 PM To: Methods@magpie.bio.indiana.edu Subject: Ampicillin Hi, I was wondering what kind of ampicillin should I use to make LB-amp+ media and LB-agar plates I searched the internet and there are three versions of amplicillins 1. ampicillin 2. ampicillin sodium salt 3. ampicillin trihydrate I am pretty sure people use ampicillin sodium salt for cell culture, just want to make sure that what I should use for bacteria culture. By the way, when these things go into water, do they have the same activity? Thanks, Wen _______________________________________________ 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 tk from mit.edu Tue Jun 10 14:36:37 2008 From: tk from mit.edu (Tom Knight) Date: Tue Jun 10 16:38:30 2008 Subject: Ampicillin References: Message-ID: Wen Huang writes: > I was wondering what kind of ampicillin should I use to make LB-amp+ > media and LB-agar plates > 2. ampicillin sodium salt This one. If you dissolve it in 50% ethanol it will remain liquid at -20, which makes adding the solution to media and agar easier. From akhan357 from sbcglobal.net Tue Jun 10 15:04:52 2008 From: akhan357 from sbcglobal.net (AK) Date: Tue Jun 10 16:38:38 2008 Subject: Inactivation of DNase I References: Message-ID: I assume you are using DNase to remove DNA from your RNA prep to be used in RT or real-time PCR. You can not heat RNA sample since 60C with divalent cations will degrade RNA. so why bother to test this. in any case, since Turbo DNase is so much more potent, perhaps a few molecules with residual activity are doing this. why not revert back to regular DNase, if that will produce sufficient chopping and subsequently can be removed/degraded completely. In the past I have used Ambion's regular DNase free and its removal with their kit (no heating involved) without any problems for RT-PCR etc. hth AK "Gouttesoulard Marina - Saint-Beauzire " wrote in message news:mailman.385.1213106791.3533.methods@net.bio.net... Hello, Have you solved your inactivation DNase problem? I have also a problem in inactivation of DNase. I inactive DNase by heating step 10min at 75°C. To check if the inactivation is efficient, I add 1ng DNA in my inactivated sample and I compare the results obtained in PCR with a control containing 1 ng DNA. There is a shift of 2 cycles with the inactivated sample. So I think that the Dnase is not totally inactivated and act on DNA added to destroy it. Have you got some solution to inactivate Dnase without damage RNA. Thanks a lot. rq : the Dnase that I used was the Turbo Dnase from ambion and I already test the inactivator of ambion's kit. Marina > mailto:marina.gouttesoulard@lonza.com > http://www.lonza.com > > This communication and its attachments, if any, may contain confidential and privileged information the use of which by other persons or entities than the intended recipient is prohibited. If you receive this transmission in error, please contact the sender immediately and delete the material from your system. From agbiok4 from gmail.com Wed Jun 11 07:52:10 2008 From: agbiok4 from gmail.com (kamalaker nasani) Date: Wed Jun 11 08:22:28 2008 Subject: Fwd: Protein work In-Reply-To: References: Message-ID: ---------- Forwarded message ---------- From: kamalaker nasani Date: Wed, Jun 11, 2008 at 6:20 PM Subject: Protein work To: methods@net.bio.net, Plant Tissue Culture Good morning, I need a protocol to enrich or to concentrate protein of specific KD from plant material. Details: I need to concentrate 10 times more of trasgene protein from seed powder. The gene is Cry1Ac. If anybody having any material related to this please share with me. -- 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 Tue Jun 10 21:43:17 2008 From: M.Dunowska from massey.ac.nz (Dunowska, Magda) Date: Wed Jun 11 08:22:36 2008 Subject: DNA isolation using Trizol Message-ID: <92FDFD8B26EB6542B1E1BF017BB998D1480CE892D6@TUR-EXCHMBX.massey.ac.nz> Hi, I=92ve been trying to isolate RNA and DNA from the same sample (tissue cult= ure fluid spiked with viruses) and, while RNA isolation seems to be working= fine, I don=92t recover any viral DNA that I originally put into the sampl= e. According to the protocol supplied with Trizol (Invitrogen), DNA should = be precipitated by ethanol and pelleted by centrifugation at 2,000 g for 5 = minutes, following several washes with the same centrifugation speed to rec= over the DNA pellet. The recommended speed of the centrifugation seems very= low, so if those of you who use Trizol could tell me whether or not they s= tick to the recommended protocol, I would be grateful=85 Thanks! Magda Magda Dunowska, LW, PhD Senior Lecturer in Veterinary Infectious Diseases (Virology) Institute of Veterinary, Animal and Biomedical Sciences Te Kura M=E2tauranga Kararehe Massey University Palmerston North New Zealand Phone : (06) 350-5799 ext 7571 Website : http://ivabs.massey.ac.nz From akhan357 from sbcglobal.net Wed Jun 11 09:54:26 2008 From: akhan357 from sbcglobal.net (AK) Date: Wed Jun 11 14:10:14 2008 Subject: Protein work References: Message-ID: how about using ultrafiltration devices sold by amicon (part of millipore now). 10 fold concentration is easily achieved by these. one example is here http://www.millipore.com/catalogue/item/42423 AK "kamalaker nasani" wrote in message news:mailman.405.1213190547.3533.methods@net.bio.net... > ---------- Forwarded message ---------- > From: kamalaker nasani > Date: Wed, Jun 11, 2008 at 6:20 PM > Subject: Protein work > To: methods@net.bio.net, Plant Tissue Culture > > > Good morning, > > > I need a protocol to enrich or to concentrate protein of specific KD from > plant material. > > Details: I need to concentrate 10 times more of trasgene protein from > seed > powder. The gene is Cry1Ac. > > If anybody having any material related to this please share with me. > > > > -- > 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 virashkgupta from gmail.com Wed Jun 11 11:15:08 2008 From: virashkgupta from gmail.com (Virash Gupta) Date: Wed Jun 11 14:10:22 2008 Subject: Methods Digest, Vol 37, Issue 8 In-Reply-To: <200806101704.m5AH4LO02125@net.bio.net> References: <200806101704.m5AH4LO02125@net.bio.net> Message-ID: For LB-Ampicillin use only Sodium Salt. Others will not be soluble in water On 6/10/08, methods-request@oat.bio.indiana.edu wrote: > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. Ampicillin (Wen Huang) > 2. Inactivation of DNase I (Gouttesoulard Marina - Saint-Beauzire ) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Mon, 09 Jun 2008 14:45:19 -0500 > From: Wen Huang > Subject: Ampicillin > To: Methods@magpie.bio.indiana.edu > Message-ID: <8BDEEB82-30A2-4B48-A871-51FE0FE33F43@ustc.edu> > Content-Type: text/plain; charset=US-ASCII; format=flowed; delsp=yes > > Hi, > > I was wondering what kind of ampicillin should I use to make LB-amp+ > media and LB-agar plates > > I searched the internet and there are three versions of amplicillins > > 1. ampicillin > 2. ampicillin sodium salt > 3. ampicillin trihydrate > > I am pretty sure people use ampicillin sodium salt for cell culture, > just want to make sure that what I should use for bacteria culture. > > By the way, when these things go into water, do they have the same > activity? > > Thanks, > Wen > > > > ------------------------------ > > Message: 2 > Date: Tue, 10 Jun 2008 15:30:13 +0200 > From: "Gouttesoulard Marina - Saint-Beauzire " > > Subject: Inactivation of DNase I > To: > Message-ID: > <2751B10B7FB0A849860E80F52E796B490362A41F@chvex24.lonzagroup.net> > Content-Type: text/plain; charset="iso-8859-1" > > Hello, > Have you solved your inactivation DNase problem? > I have also a problem in inactivation of DNase. I inactive DNase by heating step 10min at 75?C. To check if the inactivation is efficient, I add 1ng DNA in my inactivated sample and I compare the results obtained in PCR with a control containing 1 ng DNA. There is a shift of 2 cycles with the inactivated sample. So I think that the Dnase is not totally inactivated and act on DNA added to destroy it. Have you got some solution to inactivate Dnase without damage RNA. > Thanks a lot. > rq : the Dnase that I used was the Turbo Dnase from ambion and I already test the inactivator of ambion's kit. > > > Marina > > mailto:marina.gouttesoulard@lonza.com > > http://www.lonza.com > > > > > > This communication and its attachments, if any, may contain confidential and privileged information the use of > which by other persons or entities than the intended recipient is prohibited. If you receive this transmission > in error, please contact the sender immediately and delete the material from your system. > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 37, Issue 8 > ************************************** > -- Dr V K Gupta Sr Microbiologist (Molecular Biology) Insect Molecular Biology Lab Department of Entomology Punjab Agricultural University Ludhiana (Pb)-141004- India M: 09815963210 From virashkgupta from gmail.com Wed Jun 11 11:15:54 2008 From: virashkgupta from gmail.com (Virash Gupta) Date: Wed Jun 11 14:10:28 2008 Subject: Methods Digest, Vol 37, Issue 8 In-Reply-To: References: <200806101704.m5AH4LO02125@net.bio.net> Message-ID: For DNase inactivation use Heating in combination with added EDTA On 6/11/08, Virash Gupta wrote: > For LB-Ampicillin use only Sodium Salt. Others will not be soluble in water > > > On 6/10/08, methods-request@oat.bio.indiana.edu > wrote: > > Send Methods mailing list submissions to > > methods@net.bio.net > > > > To subscribe or unsubscribe via the World Wide Web, visit > > http://www.bio.net/biomail/listinfo/methods > > or, via email, send a message with subject or body 'help' to > > methods-request@net.bio.net > > > > You can reach the person managing the list at > > methods-owner@net.bio.net > > > > When replying, please edit your Subject line so it is more specific > > than "Re: Contents of Methods digest..." > > > > > > Today's Topics: > > > > 1. Ampicillin (Wen Huang) > > 2. Inactivation of DNase I (Gouttesoulard Marina - Saint-Beauzire ) > > > > > > ---------------------------------------------------------------------- > > > > Message: 1 > > Date: Mon, 09 Jun 2008 14:45:19 -0500 > > From: Wen Huang > > Subject: Ampicillin > > To: Methods@magpie.bio.indiana.edu > > Message-ID: <8BDEEB82-30A2-4B48-A871-51FE0FE33F43@ustc.edu> > > Content-Type: text/plain; charset=US-ASCII; format=flowed; delsp=yes > > > > Hi, > > > > I was wondering what kind of ampicillin should I use to make LB-amp+ > > media and LB-agar plates > > > > I searched the internet and there are three versions of amplicillins > > > > 1. ampicillin > > 2. ampicillin sodium salt > > 3. ampicillin trihydrate > > > > I am pretty sure people use ampicillin sodium salt for cell culture, > > just want to make sure that what I should use for bacteria culture. > > > > By the way, when these things go into water, do they have the same > > activity? > > > > Thanks, > > Wen > > > > > > > > ------------------------------ > > > > Message: 2 > > Date: Tue, 10 Jun 2008 15:30:13 +0200 > > From: "Gouttesoulard Marina - Saint-Beauzire " > > > > Subject: Inactivation of DNase I > > To: > > Message-ID: > > <2751B10B7FB0A849860E80F52E796B490362A41F@chvex24.lonzagroup.net> > > Content-Type: text/plain; charset="iso-8859-1" > > > > Hello, > > Have you solved your inactivation DNase problem? > > I have also a problem in inactivation of DNase. I inactive DNase by heating step 10min at 75?C. To check if the inactivation is efficient, I add 1ng DNA in my inactivated sample and I compare the results obtained in PCR with a control containing 1 ng DNA. There is a shift of 2 cycles with the inactivated sample. So I think that the Dnase is not totally inactivated and act on DNA added to destroy it. Have you got some solution to inactivate Dnase without damage RNA. > > Thanks a lot. > > rq : the Dnase that I used was the Turbo Dnase from ambion and I already test the inactivator of ambion's kit. > > > > > > Marina > > > mailto:marina.gouttesoulard@lonza.com > > > http://www.lonza.com > > > > > > > > > > This communication and its attachments, if any, may contain confidential and privileged information the use of > > which by other persons or entities than the intended recipient is prohibited. If you receive this transmission > > in error, please contact the sender immediately and delete the material from your system. > > > > > > ------------------------------ > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > End of Methods Digest, Vol 37, Issue 8 > > ************************************** > > > > > -- > Dr V K Gupta > Sr Microbiologist (Molecular Biology) > Insect Molecular Biology Lab > Department of Entomology > Punjab Agricultural University > Ludhiana (Pb)-141004- India > M: 09815963210 > -- Dr V K Gupta Sr Microbiologist (Molecular Biology) Insect Molecular Biology Lab Department of Entomology Punjab Agricultural University Ludhiana (Pb)-141004- India M: 09815963210 From sudhee26 from gmail.com Thu Jun 12 13:17:37 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu Jun 12 16:33:04 2008 Subject: DNA isolation using Trizol In-Reply-To: References: <92FDFD8B26EB6542B1E1BF017BB998D1480CE892D6@TUR-EXCHMBX.massey.ac.nz> Message-ID: aSB0aGluayBpbmN1YmF0aW9uIGluIHNvZGl1bSBjaXRyYXRlIHN0ZXAgaXMgdGhlIGtleSB0byBn ZXQgZ29vZCB5aWVsZC4KCjIwMDgvNi8xMiBTdWRoZWVuZHJhIFJhbyBOIFIgPHN1ZGhlZTI2QGdt YWlsLmNvbT46Cgo+IEhpLAo+IGkgaGF2ZSBpc29sYXRlZCBETkEgZnJvbSBUcml6b2wgcmVhZ2Vu dCAoY2VsbHMpLi5pdCBhbHNvIGludm9sdmVzIGEgc3RlcCBvZgo+IHNvZGl1bSBjaXRyYXRlLCAo cmVmIHRyaXpvbCBtYW51YWwgZm9yIGRldGFpbHMpIGluY3ViYXRpb24gZXRjLCBub3QganVzdAo+ IGNlbnRyaWZ1Z2F0aW9uIGF0IGxvdyBzcGVlZHMgaWYgaSBhbSBub3Qgd3JvbmcuCj4gbm8gaWRl YSBhYm91dCB2aXJhbCBETkEgdGhvdWdoLi5pIHRoaW5rIGl0IHNob3VsZCBub3QgYmUgZGlmZmVy ZW50Li4KPgo+IFN1ZGhlZW5kcmEuCj4KPgo+Cj4gMjAwOC82LzExIER1bm93c2thLCBNYWdkYSA8 TS5EdW5vd3NrYUBtYXNzZXkuYWMubno+Ogo+Cj4gSGksCj4+IEkndmUgYmVlbiB0cnlpbmcgdG8g aXNvbGF0ZSBSTkEgYW5kIEROQSBmcm9tIHRoZSBzYW1lIHNhbXBsZSAodGlzc3VlCj4+IGN1bHR1 cmUgZmx1aWQgc3Bpa2VkIHdpdGggdmlydXNlcykgYW5kLCB3aGlsZSBSTkEgaXNvbGF0aW9uIHNl ZW1zIHRvIGJlCj4+IHdvcmtpbmcgZmluZSwgSSBkb24ndCByZWNvdmVyIGFueSB2aXJhbCBETkEg dGhhdCBJIG9yaWdpbmFsbHkgcHV0IGludG8gdGhlCj4+IHNhbXBsZS4gQWNjb3JkaW5nIHRvIHRo ZSBwcm90b2NvbCBzdXBwbGllZCB3aXRoIFRyaXpvbCAoSW52aXRyb2dlbiksIEROQQo+PiBzaG91 bGQgYmUgcHJlY2lwaXRhdGVkIGJ5IGV0aGFub2wgYW5kIHBlbGxldGVkIGJ5IGNlbnRyaWZ1Z2F0 aW9uIGF0IDIsMDAwIGcKPj4gZm9yIDUgbWludXRlcywgZm9sbG93aW5nIHNldmVyYWwgd2FzaGVz IHdpdGggdGhlIHNhbWUgY2VudHJpZnVnYXRpb24gc3BlZWQKPj4gdG8gcmVjb3ZlciB0aGUgRE5B IHBlbGxldC4gVGhlIHJlY29tbWVuZGVkIHNwZWVkIG9mIHRoZSBjZW50cmlmdWdhdGlvbiBzZWVt cwo+PiB2ZXJ5IGxvdywgc28gaWYgdGhvc2Ugb2YgeW91IHdobyB1c2UgVHJpem9sIGNvdWxkIHRl bGwgbWUgd2hldGhlciBvciBub3QKPj4gdGhleSBzdGljayB0byB0aGUgcmVjb21tZW5kZWQgcHJv dG9jb2wsIEkgd291bGQgYmUgZ3JhdGVmdWyhrQo+PiBUaGFua3MhCj4+IE1hZ2RhCj4+Cj4+Cj4+ IE1hZ2RhIER1bm93c2thLCBMVywgUGhECj4+Cj4+IFNlbmlvciBMZWN0dXJlciBpbiBWZXRlcmlu YXJ5IEluZmVjdGlvdXMgRGlzZWFzZXMgKFZpcm9sb2d5KQo+Pgo+PiBJbnN0aXR1dGUgb2YgVmV0 ZXJpbmFyeSwgQW5pbWFsIGFuZCBCaW9tZWRpY2FsIFNjaWVuY2VzCj4+Cj4+IFRlIEt1cmEgTaih dGF1cmFuZ2EgS2FyYXJlaGUKPj4KPj4gTWFzc2V5IFVuaXZlcnNpdHkKPj4KPj4gUGFsbWVyc3Rv biBOb3J0aAo+Pgo+PiBOZXcgWmVhbGFuZAo+Pgo+Pgo+Pgo+PiBQaG9uZSA6ICgwNikgMzUwLTU3 OTkgZXh0IDc1NzEKPj4KPj4gV2Vic2l0ZSA6IGh0dHA6Ly9pdmFicy5tYXNzZXkuYWMubno8aHR0 cDovL2l2YWJzLm1hc3NleS5hYy5uei8+Cj4+Cj4+IF9fX19fX19fX19fX19fX19fX19fX19fX19f X19fX19fX19fX19fX19fX19fX19fCj4+IE1ldGhvZHMgbWFpbGluZyBsaXN0Cj4+IE1ldGhvZHNA bmV0LmJpby5uZXQKPj4gaHR0cDovL3d3dy5iaW8ubmV0L2Jpb21haWwvbGlzdGluZm8vbWV0aG9k cwo+Pgo+Cj4KPgo+IC0tCj4gVGhpbmsgYmVmb3JlIGFncmVlCj4gVGhpbmsgYmVmb3JlIHlvdSBu b2QKPiBidXQgU1RPUCB0aGlua2luZwo+IGFuZCBZb3UgQXJlIEdvZAoKCgoKLS0gClRoaW5rIGJl Zm9yZSBhZ3JlZQpUaGluayBiZWZvcmUgeW91IG5vZApidXQgU1RPUCB0aGlua2luZwphbmQgWW91 IEFyZSBHb2QKFrom evan.g.reed from gmail.com Thu Jun 12 16:56:35 2008 From: evan.g.reed from gmail.com (Evan Reed) Date: Thu Jun 12 18:03:45 2008 Subject: Poor resolution when isolating ssDNA? Message-ID: <039DEDB2-D81D-4249-8B8B-F00C38AE7765@gmail.com> Hej, I am trying to isolate ssDNA after binding dsDNA oligos with biotinylated antisense strands to strep microbeads, denaturing with NaOH, then adding the supernatant containing the sense ssDNA to a NAP-5 sephadex desalting column. Unfortunately, I am getting terrible resolution when I try to elute on the column -- even after 2 mL of elution, I am still getting detectable levels of ssDNA in the eluate (dsDNA oligos of the same length elute just fine in 0.5 mL, with acceptable resolution). The amount of DNA loaded into the column should be well below the binding capacity so I am not sure why this is happening. Has anyone done this before? Is there a better way to purify the ssDNA instead of this column? Thanks, ER From virashkgupta from gmail.com Fri Jun 13 02:17:38 2008 From: virashkgupta from gmail.com (Virash Gupta) Date: Fri Jun 13 11:01:01 2008 Subject: Methods Digest, Vol 37, Issue 10 In-Reply-To: <200806121704.m5CH4XO02182@net.bio.net> References: <200806121704.m5CH4XO02182@net.bio.net> Message-ID: concentarting Cry1Ac from Bt-plant is not easy as it is truncated protein. better and easy to produce recombinant synthetic cry1Ac protein using cloned genes available from dr Altosaar or others and cRy1Ac specific antibodies. On 6/12/08, methods-request@oat.bio.indiana.edu wrote: > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. Re: Protein work (AK) > 2. Re: Methods Digest, Vol 37, Issue 8 (Virash Gupta) > 3. Re: Methods Digest, Vol 37, Issue 8 (Virash Gupta) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 11 Jun 2008 09:54:26 -0500 > From: "AK" > Subject: Re: Protein work > To: methods@net.bio.net > Message-ID: > > how about using ultrafiltration devices sold by amicon (part of millipore > now). 10 fold concentration is easily achieved by these. one example is here > http://www.millipore.com/catalogue/item/42423 > AK > > "kamalaker nasani" wrote in message > news:mailman.405.1213190547.3533.methods@net.bio.net... > > ---------- Forwarded message ---------- > > From: kamalaker nasani > > Date: Wed, Jun 11, 2008 at 6:20 PM > > Subject: Protein work > > To: methods@net.bio.net, Plant Tissue Culture > > > > > > Good morning, > > > > > > I need a protocol to enrich or to concentrate protein of specific KD from > > plant material. > > > > Details: I need to concentrate 10 times more of trasgene protein from > > seed > > powder. The gene is Cry1Ac. > > > > If anybody having any material related to this please share with me. > > > > > > > > -- > > 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 > > > > > ------------------------------ > > Message: 2 > Date: Wed, 11 Jun 2008 21:45:08 +0530 > From: "Virash Gupta" > Subject: Re: Methods Digest, Vol 37, Issue 8 > To: methods@oat.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > For LB-Ampicillin use only Sodium Salt. Others will not be soluble in water > > > On 6/10/08, methods-request@oat.bio.indiana.edu > wrote: > > Send Methods mailing list submissions to > > methods@net.bio.net > > > > To subscribe or unsubscribe via the World Wide Web, visit > > http://www.bio.net/biomail/listinfo/methods > > or, via email, send a message with subject or body 'help' to > > methods-request@net.bio.net > > > > You can reach the person managing the list at > > methods-owner@net.bio.net > > > > When replying, please edit your Subject line so it is more specific > > than "Re: Contents of Methods digest..." > > > > > > Today's Topics: > > > > 1. Ampicillin (Wen Huang) > > 2. Inactivation of DNase I (Gouttesoulard Marina - Saint-Beauzire ) > > > > > > ---------------------------------------------------------------------- > > > > Message: 1 > > Date: Mon, 09 Jun 2008 14:45:19 -0500 > > From: Wen Huang > > Subject: Ampicillin > > To: Methods@magpie.bio.indiana.edu > > Message-ID: <8BDEEB82-30A2-4B48-A871-51FE0FE33F43@ustc.edu> > > Content-Type: text/plain; charset=US-ASCII; format=flowed; delsp=yes > > > > Hi, > > > > I was wondering what kind of ampicillin should I use to make LB-amp+ > > media and LB-agar plates > > > > I searched the internet and there are three versions of amplicillins > > > > 1. ampicillin > > 2. ampicillin sodium salt > > 3. ampicillin trihydrate > > > > I am pretty sure people use ampicillin sodium salt for cell culture, > > just want to make sure that what I should use for bacteria culture. > > > > By the way, when these things go into water, do they have the same > > activity? > > > > Thanks, > > Wen > > > > > > > > ------------------------------ > > > > Message: 2 > > Date: Tue, 10 Jun 2008 15:30:13 +0200 > > From: "Gouttesoulard Marina - Saint-Beauzire " > > > > Subject: Inactivation of DNase I > > To: > > Message-ID: > > <2751B10B7FB0A849860E80F52E796B490362A41F@chvex24.lonzagroup.net> > > Content-Type: text/plain; charset="iso-8859-1" > > > > Hello, > > Have you solved your inactivation DNase problem? > > I have also a problem in inactivation of DNase. I inactive DNase by heating step 10min at 75?C. To check if the inactivation is efficient, I add 1ng DNA in my inactivated sample and I compare the results obtained in PCR with a control containing 1 ng DNA. There is a shift of 2 cycles with the inactivated sample. So I think that the Dnase is not totally inactivated and act on DNA added to destroy it. Have you got some solution to inactivate Dnase without damage RNA. > > Thanks a lot. > > rq : the Dnase that I used was the Turbo Dnase from ambion and I already test the inactivator of ambion's kit. > > > > > > Marina > > > mailto:marina.gouttesoulard@lonza.com > > > http://www.lonza.com > > > > > > > > > > This communication and its attachments, if any, may contain confidential and privileged information the use of > > which by other persons or entities than the intended recipient is prohibited. If you receive this transmission > > in error, please contact the sender immediately and delete the material from your system. > > > > > > ------------------------------ > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > End of Methods Digest, Vol 37, Issue 8 > > ************************************** > > > > > -- > Dr V K Gupta > Sr Microbiologist (Molecular Biology) > Insect Molecular Biology Lab > Department of Entomology > Punjab Agricultural University > Ludhiana (Pb)-141004- India > M: 09815963210 > > > > ------------------------------ > > Message: 3 > Date: Wed, 11 Jun 2008 21:45:54 +0530 > From: "Virash Gupta" > Subject: Re: Methods Digest, Vol 37, Issue 8 > To: methods@oat.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > For DNase inactivation use Heating in combination with added EDTA > > > On 6/11/08, Virash Gupta wrote: > > For LB-Ampicillin use only Sodium Salt. Others will not be soluble in water > > > > > > On 6/10/08, methods-request@oat.bio.indiana.edu > > wrote: > > > Send Methods mailing list submissions to > > > methods@net.bio.net > > > > > > To subscribe or unsubscribe via the World Wide Web, visit > > > http://www.bio.net/biomail/listinfo/methods > > > or, via email, send a message with subject or body 'help' to > > > methods-request@net.bio.net > > > > > > You can reach the person managing the list at > > > methods-owner@net.bio.net > > > > > > When replying, please edit your Subject line so it is more specific > > > than "Re: Contents of Methods digest..." > > > > > > > > > Today's Topics: > > > > > > 1. Ampicillin (Wen Huang) > > > 2. Inactivation of DNase I (Gouttesoulard Marina - Saint-Beauzire ) > > > > > > > > > ---------------------------------------------------------------------- > > > > > > Message: 1 > > > Date: Mon, 09 Jun 2008 14:45:19 -0500 > > > From: Wen Huang > > > Subject: Ampicillin > > > To: Methods@magpie.bio.indiana.edu > > > Message-ID: <8BDEEB82-30A2-4B48-A871-51FE0FE33F43@ustc.edu> > > > Content-Type: text/plain; charset=US-ASCII; format=flowed; delsp=yes > > > > > > Hi, > > > > > > I was wondering what kind of ampicillin should I use to make LB-amp+ > > > media and LB-agar plates > > > > > > I searched the internet and there are three versions of amplicillins > > > > > > 1. ampicillin > > > 2. ampicillin sodium salt > > > 3. ampicillin trihydrate > > > > > > I am pretty sure people use ampicillin sodium salt for cell culture, > > > just want to make sure that what I should use for bacteria culture. > > > > > > By the way, when these things go into water, do they have the same > > > activity? > > > > > > Thanks, > > > Wen > > > > > > > > > > > > ------------------------------ > > > > > > Message: 2 > > > Date: Tue, 10 Jun 2008 15:30:13 +0200 > > > From: "Gouttesoulard Marina - Saint-Beauzire " > > > > > > Subject: Inactivation of DNase I > > > To: > > > Message-ID: > > > <2751B10B7FB0A849860E80F52E796B490362A41F@chvex24.lonzagroup.net> > > > Content-Type: text/plain; charset="iso-8859-1" > > > > > > Hello, > > > Have you solved your inactivation DNase problem? > > > I have also a problem in inactivation of DNase. I inactive DNase by heating step 10min at 75?C. To check if the inactivation is efficient, I add 1ng DNA in my inactivated sample and I compare the results obtained in PCR with a control containing 1 ng DNA. There is a shift of 2 cycles with the inactivated sample. So I think that the Dnase is not totally inactivated and act on DNA added to destroy it. Have you got some solution to inactivate Dnase without damage RNA. > > > Thanks a lot. > > > rq : the Dnase that I used was the Turbo Dnase from ambion and I already test the inactivator of ambion's kit. > > > > > > > > > Marina > > > > mailto:marina.gouttesoulard@lonza.com > > > > http://www.lonza.com > > > > > > > > > > > > > > This communication and its attachments, if any, may contain confidential and privileged information the use of > > > which by other persons or entities than the intended recipient is prohibited. If you receive this transmission > > > in error, please contact the sender immediately and delete the material from your system. > > > > > > > > > ------------------------------ > > > > > > _______________________________________________ > > > Methods mailing list > > > Methods@net.bio.net > > > http://www.bio.net/biomail/listinfo/methods > > > > > > End of Methods Digest, Vol 37, Issue 8 > > > ************************************** > > > > > > > > > -- > > Dr V K Gupta > > Sr Microbiologist (Molecular Biology) > > Insect Molecular Biology Lab > > Department of Entomology > > Punjab Agricultural University > > Ludhiana (Pb)-141004- India > > M: 09815963210 > > > > > -- > Dr V K Gupta > Sr Microbiologist (Molecular Biology) > Insect Molecular Biology Lab > Department of Entomology > Punjab Agricultural University > Ludhiana (Pb)-141004- India > M: 09815963210 > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 37, Issue 10 > *************************************** > -- Dr V K Gupta Sr Microbiologist (Molecular Biology) Insect Molecular Biology Lab Department of Entomology Punjab Agricultural University Ludhiana (Pb)-141004- India M: 09815963210 From peter.ianakiev from gmail.com Fri Jun 13 07:33:13 2008 From: peter.ianakiev from gmail.com (peter) Date: Fri Jun 13 11:01:07 2008 Subject: Pfu question Message-ID: <91223fdf-0cbb-410c-a32e-9f57e983068e@x41g2000hsb.googlegroups.com> Hi guys, Does anyone knows if Pfu leaves even small percentage of A tails on the PCR product? Thanks, Peter From pumituki from hotmail.com Fri Jun 13 09:58:45 2008 From: pumituki from hotmail.com (Maria Labandeira-Rey) Date: Fri Jun 13 11:01:13 2008 Subject: RNA extraction Message-ID: I have been told that I have to add sodium azide to my bacterial cultures right before the RNA extraction to prevent RNA degradation. I would like to know if anyone knows the basis for this degradation inhibition. In my previous lab we just pelleted and re-suspended in trizol. Thanks in advance. _________________________________________________________________ Enjoy 5 GB of free, password-protected online storage. http://www.windowslive.com/skydrive/overview.html?ocid=TXT_TAGLM_WL_Refresh_skydrive_062008 From josemhoyos from hotmail.com Fri Jun 13 03:27:47 2008 From: josemhoyos from hotmail.com (josefina martinez) Date: Fri Jun 13 11:03:07 2008 Subject: pPNT Message-ID: Hi, My name is Josefina Martinez Hoyos. I would like to ask you if you found the sequence of pPNT vector, because I am also interested in it. Waiting for an answer, Josefina _________________________________________________________________ MSN Video. http://video.msn.com/?mkt=es-es From yulia24 from gmail.com Fri Jun 13 12:09:24 2008 From: yulia24 from gmail.com (Yulia Shifrin) Date: Fri Jun 13 13:01:40 2008 Subject: pPNT In-Reply-To: References: Message-ID: <9c7f9b9b0806131009u1ce81200oa7ef507bb2b8f1c9@mail.gmail.com> I found this link: http://www.med.umich.edu/tamc/mta.html#pPNT Hope it's the one you are looking for. Yulia On Fri, Jun 13, 2008 at 4:27 AM, josefina martinez wrote: > > > Hi, > > My name is Josefina Martinez Hoyos. I would like to ask you if you found > the sequence of pPNT vector, because I am also interested in it. > > Waiting for an answer, > > Josefina > _________________________________________________________________ > MSN Video. > http://video.msn.com/?mkt=es-es > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From akhan357 from sbcglobal.net Fri Jun 13 18:03:28 2008 From: akhan357 from sbcglobal.net (AK) Date: Fri Jun 13 22:42:19 2008 Subject: RNA extraction References: Message-ID: <81D4k.5053$N87.1695@nlpi068.nbdc.sbc.com> Let me take a guess, increased proteolysis or decreased protein synthesis or both? Let us all know the correct answer to this interesting question, if you find it. In your previous lab were you using the same bugs? apparently people chill or azide treat the bacteria before RNA prep. Also, it seems that azide only works against gram negative bacteria! AK "Maria Labandeira-Rey" wrote in message news:mailman.433.1213372918.3533.methods@net.bio.net... I have been told that I have to add sodium azide to my bacterial cultures right before the RNA extraction to prevent RNA degradation. I would like to know if anyone knows the basis for this degradation inhibition. In my previous lab we just pelleted and re-suspended in trizol. Thanks in advance. _________________________________________________________________ Enjoy 5 GB of free, password-protected online storage. http://www.windowslive.com/skydrive/overview.html?ocid=TXT_TAGLM_WL_Refresh_skydrive_062008= From rijutak from googlemail.com Mon Jun 16 03:46:29 2008 From: rijutak from googlemail.com (rijuta kotenkar) Date: Mon Jun 16 11:35:02 2008 Subject: Pfu question Message-ID: <71d8f0270806160146u5ae051bfte4657b7f3234b8bb@mail.gmail.com> Hi, As my experience goes i have had no problems with Pfu polymerase for creating blunt ends. But it is very slow compared to Taq polymerase and sometimes can generate truncated products due to its high 3' to 5' exonuclease proofreading activity. RK > Message: 3 > Date: Fri, 13 Jun 2008 05:33:13 -0700 (PDT) > From: peter > Subject: Pfu question > To: methods@net.bio.net > Message-ID: > <91223fdf-0cbb-410c-a32e-9f57e983068e@x41g2000hsb.googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Hi guys, > Does anyone knows if Pfu leaves even small percentage of A tails on > the PCR product? Thanks, > Peter > > > From cv2135 from columbia.edu Mon Jun 16 09:15:37 2008 From: cv2135 from columbia.edu (cv2135@columbia.edu) Date: Mon Jun 16 11:35:26 2008 Subject: Non-isotopic method for In vitro transcription Message-ID: <20080616101537.rgczua2fr4w0080c@cubmail.cc.columbia.edu> Hello all Apparently the price of P32 has become prohibitive (4 X as expensive as previously). Does anyone know of an alternative, non isotropic method that we can use for IVT labelling? thanks chris From cheetah.zao from gmail.com Tue Jun 17 01:12:20 2008 From: cheetah.zao from gmail.com (cheetah.zao@gmail.com) Date: Tue Jun 17 12:34:16 2008 Subject: Poor resolution when isolating ssDNA? References: Message-ID: <5145efed-bf2d-45bd-be7d-f91b3dbca297@w1g2000prd.googlegroups.com> 70% enthenol precipitation in the presence of 50 mM MgCl2 may work. On Jun 13, 5:56 am, Evan Reed wrote: > Hej, > > I am trying to isolate ssDNA after binding dsDNA oligos with > biotinylated antisense strands to strep microbeads, denaturing with > NaOH, then adding the supernatant containing the sense ssDNA to a > NAP-5 sephadex desalting column. Unfortunately, I am getting terrible > resolution when I try to elute on the column -- even after 2 mL of > elution, I am still getting detectable levels of ssDNA in the eluate > (dsDNA oligos of the same length elute just fine in 0.5 mL, with > acceptable resolution). The amount of DNA loaded into the column > should be well below the binding capacity so I am not sure why this is > happening. Has anyone done this before? Is there a better way to > purify the ssDNA instead of this column? > > Thanks, > > ER From pjie2 from cam.ac.uk Tue Jun 17 06:06:12 2008 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Tue Jun 17 12:34:43 2008 Subject: Advice needed for Western blotting Message-ID: Hiya, I am just starting up a project investigating two novel proteins in testis function. My background is in molecular genetics and transcription profiling, so protein work is all pretty new to me. We have peptide antibodies prepared (or in the works) for both proteins - the first step is obviously to characterise the antibodies. We're using peptide antibodies rather than antibodies to recombinant protein because there are many related genes, and we want to avoid cross-reactivity. We have ORFs cloned for each of the genes and their close relatives. The aim is to use in vitro transcription/translation to generate pure proteins in order to check the antibody specificity. Once we know that the antibodies do actually detect our proteins of interest (and not the relatives), we can move on to other more interesting questions! So the first plan is Western blots using whole testis extracts to see whether we have a band of the expected size in testis. We'll then do further Western blots of testis extract alongside the various in vitro translated proteins in order to check AB specificity. At this point I'm looking for advice / sanity checking on how to extract my proteins and what sort of gels to use for the Western blots. On looking around in the literature, I'm finding myself a little bewildered by buffer compositions - many papers refer to "standard RIPA buffer" or "standard Laemmli sample buffer", and yet there seem to be as many different recipes as there are laboratories. Secondly, it looks like the most common buffers simply won't work for my proteins! Details below for each of the proteins of interest: ************************************************* Protein 1: H2AL1 ---------------- This one is a novel histone. I've looked at the QIAGEN QProteome Nuclear kit, but I'm loath to commit to using that as it doesn't tell you what any of the buffers are. I can't even tell if it's an acid extraction or a salt extraction! Also, it's very expensive for the amount of tissue you can process with it. So I'd prefer to do my own histone extraction if possible. I believe that means I want to prepare purified nuclei and then do an acid extraction. Previously I've done nuclear preps when preparing high molecular weight gDNA - will the same buffers be appropriate for protein work? Protocol is as follows: * Homogenise tissue at low speed in lysis buffer [0.05M TrisCl pH 7.5, 1mM EDTA, 5mM MgCl2, 50mM NaCl, 5% glycerol, 1% Triton X-100, 1% B-ME], using 100 mg tissue / ml buffer. * Centrifuge to spin down nuclei * Wash in lysis buffer and spin down again Once I've got the purified nuclei, the histone prep looks admirably straightforward. According to Abcam, you just resuspend the nuclear pellet in 0.2M HCl and leave it overnight at 4 degrees C. Centrifuge to spin down debris and keep the supernatant. However, other protocols seem to use different acid concentrations, or H2SO4 instead of HCl, and may even include B-ME in the histone extraction buffer. One protocol I Googled called for you to neutralise the preparation with KOH afterwards - is that really called for? Wouldn't it just drop the proteins straight back out of solution again? Once I've got my histone prep, what buffer should I use for running them on an SDS-PAGE gel? Standard Laemmli sample buffer? If so, *which* standard Laemmli buffer? Will a normal SDS-PAGE gel work OK, or will there be pH issues due to the sample being in pretty much neat acid? Generally the lab uses Novex gels in a XCell mini-blot module. For a histone (size ~17 kDa), it'll be quite a high percentage gel. ************************************************* Protein 1: mgclh ------------------ Localisation of this protein is unknown, but a closely related gene (Gmcl1) is known to be a nuclear lamina protein. Gmcl1 is RIPA-insoluble, so it's likely mgclh will also be RIPA-insoluble. Protein size is expected to be around 55 kDa. The plan here is to do a standard protein extraction by homogenising testis tissue in RIPA + protease inhibitors. Retain the supernatant (to get the RIPA-soluble fraction), and then dissolve the pellet in something (to get the RIPA-insoluble fraction). Question is what to dissolve the pellet in. The original literature on Gmcl1 said they resuspended the RIPA-insoluble pellet in "SDS-polyacrylamide gel electrophoresis sample buffer", but doesn't give the composition. I would assume this is 1x Laemmli sample buffer. Does this sound plausible? If so, what recipe for Laemmli buffer would you recommend? I've found half a dozen different recipes, and the only common factor between them is ~60-100mM Tris pH 6.8 and 2% SDS. There are variants with and without DTT, with and without B-ME, with and without loading dye, using sucrose or glycerol for increased density! I would imagine I'll need to add B-ME to reduce the proteins before running on the gel, but should I add the B-ME at the point I resuspend the pellet, or only when I'm about to run an aliquot on a gel? Will I need to add protease inhibitors, or will the Laemmli buffer itself take care of that? ************************************************* Thank you for your time and patience - sorry for asking so many questions, most of which will undoubtedly have obvious answers. Probably several mutually incompatible obvious answers, mind you, but that's biology for you. My theory is that it's better to ask silly questions and look daft than to forge ahead without asking and cock something up! Thanks, Peter Ellis From peter.ianakiev from gmail.com Tue Jun 17 13:19:15 2008 From: peter.ianakiev from gmail.com (peter) Date: Tue Jun 17 15:23:01 2008 Subject: T4 PNK Message-ID: <28b39b3f-6f52-4c5e-baa3-37a22181d61c@8g2000hse.googlegroups.com> Hi all, have kinda silly question: Can T4PNK use dATP instead of rATP to phosphorilate DNA? Thanks , Peter From engelbert_buxbaum from hotmail.com Tue Jun 17 14:30:32 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Jun 17 15:23:07 2008 Subject: request for papers References: <715ec031-a920-4749-b514-429b695fb1fb@v26g2000prm.googlegroups.com> Message-ID: Am 12.06.2008, 04:53 Uhr, schrieb sheelu : > Dear all friends: > I have found two papers published in Nature & Science, the name > of which > are "How biotech can transform biofuels".(Nature biotechnology (2005 > feb ) 26(2), 169-172; and another one is > "Fuel ethanol from cellulosic biomass" (Science 1991Mar 15:251(4999): > 1318-1323, I think > this paper would be great help to me.So anyone of here can help me to > get it? One method is to go to your library. If they do not have it, they can order a copy for you by interlibrary loan (not cheap though). The second method is to contact the authors and ask for a reprint. Their affiliation you get from the usual search tools, e.g. scholars.google.com or PubMed. Use email and you usually get an electronic copy within a few days. Exchange of reprints is a time-honored way of scholars to keep in contact. From engelbert_buxbaum from hotmail.com Tue Jun 17 16:41:45 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Jun 18 11:36:37 2008 Subject: Advice needed for Western blotting References: Message-ID: Am 17.06.2008, 07:06 Uhr, schrieb Peter Ellis : > We have peptide antibodies prepared (or in the works) for both proteins > - the first step is obviously to characterise the antibodies. We're > using peptide antibodies rather than antibodies to recombinant protein > because there are many related genes, and we want to avoid > cross-reactivity. That is fine, just remember that the epitope may be hidden in native proteins, thus the AB may not work e.g. in immunoprecipitation. In Western you deal with denatured proteins, so that is not a problem. > We have ORFs cloned for each of the genes and their close relatives. > The aim is to use in vitro transcription/translation to generate pure > proteins in order to check the antibody specificity. Once we know that > the antibodies do actually detect our proteins of interest (and not the > relatives), we can move on to other more interesting questions! In addition to testing against individual proteins you should test against a crude tissue lysate. You should see only a single band. Even more telling would be a blot from a 2D-gel (SDS-PAGE after IEF). > At this point I'm looking for advice / sanity checking on how to > extract my proteins and what sort of gels to use for the Western blots. The most common method is SDS-PAGE according to Laemmli (Laemmli: Nature 227 (1970) 680 and Laemmli & Favre, J. Mol. Biol. 80 (1973) 575-599). Separation is by molecular mass. For more specific detection, this is preceeded by isoelectric focussing, a method that separates by isoelectric point. See J. Klose and U. Kobalz: Electrophoresis 16 (1995) 1034-1059 > On looking around in the literature, I'm finding myself a little > bewildered by buffer compositions - many papers refer to "standard RIPA > buffer" or "standard Laemmli sample buffer", and yet there seem to be as > many different recipes as there are laboratories. Secondly, it looks > like the most common buffers simply won't work for my proteins! It's not as bad as that. The Laemmli buffer is a fairly good bet. > This one is a novel histone. I've looked at the QIAGEN QProteome > Nuclear kit, but I'm loath to commit to using that as it doesn't tell > you what any of the buffers are. I can't even tell if it's an acid > extraction or a salt extraction! Also, it's very expensive for the > amount of tissue you can process with it. Histones have a high positive charge, to get a reasonable estimate of their molecular mass from PAGE, use the positive detergent CTAB rather than the negatively charged SDS (E. Buxbaum: Anal. Biochem. 314 {2003} 70-76) > So I'd prefer to do my own histone extraction if possible. I believe > that means I want to prepare purified nuclei and then do an acid > extraction. Previously I've done nuclear preps when preparing high > molecular weight gDNA - will the same buffers be appropriate for protein > work? Protocol is as follows: > > Homogenise tissue at low speed in lysis buffer [0.05M TrisCl pH 7.5, 1mM > EDTA, 5mM MgCl2, 50mM NaCl, 5% glycerol, 1% Triton X-100, 1% B-ME], > using 100 mg tissue / ml buffer. I'd probably use KCl rather than NaCl, as the cell interior is high in K and low in Na. I'd also reduce [Mg] to 1 mM or so to discourage metal-dependent proteases. The Triton I'd leave out at this step and reduce bME to 1 mM (or replace it with DTT, which has a more appropriate redox potential). You'll need a protease inhibitor coctail (EDTA, PMSF, Pepstatin, Leupeptin, e-aminocaproic acid). > Once I've got the purified nuclei, the histone prep looks admirably > straightforward. According to Abcam, you just resuspend the nuclear > pellet in 0.2M HCl and leave it overnight at 4 degrees C. Centrifuge to > spin down debris and keep the supernatant. However, other protocols > seem to use different acid concentrations, or H2SO4 instead of HCl, and > may even include B-ME in the histone extraction buffer. The acid is quite irrelevant, the idea is simply to increase the solubility of the highly charged histons and to pellet as many of the other proteins as possible. The bME protects cysteine -SH groups from oxidation by air. However, this extraction procedure is too selective for your purposes, you will not know whether there are cross-reacting proteins in the pellet. > > One protocol I Googled called for you to neutralise the preparation with > KOH afterwards - is that really called for? Wouldn't it just drop the > proteins straight back out of solution again? No. But you need a pH near neutral and as low an ion concentration as possible for the electrophoresis step. Therefore solubilisation in sample buffer rather than HCl is better. That also gives you an idea about the cross-reactions of your antibody. > For a histone (size ~17 kDa), it'll be quite a high percentage gel. I find gradient gels (5-15 or 20%) most convenient > > Protein 1: mgclh > ------------------ > > RIPA-insoluble, so it's likely mgclh will also be RIPA-insoluble. > Protein size is expected to be around 55 kDa. Again the use of CTAB electrophoresis may help, as CTAB can solubilise proteins very efficiently. > The original literature on Gmcl1 said they resuspended the > RIPA-insoluble pellet in "SDS-polyacrylamide gel electrophoresis sample > buffer", but doesn't give the composition. I would assume this is 1x > Laemmli sample buffer. Does this sound plausible? If so, what recipe > for Laemmli buffer would you recommend? I've found half a dozen > different recipes, and the only common factor between them is ~60-100mM > Tris pH 6.8 and 2% SDS. There are variants with and without DTT, with > and without B-ME, with and without loading dye, using sucrose or > glycerol for increased density! The buffer is actually double concentrated, once you mix it with an equal volume of sample you get the working strenght. DTT and bME reduce disulphide bonds and thus lead to more complete unfolding of the proteins. DTT (Clelands reagent) smells less and has a better standard redox potential for this purpose. However, it is more expensive than bME. Wether you use glycerol or sucrose to increase density really doesn't matter, you just need either for proper gel loading, its physics, not chemistry. The dye is important not only to aid in loading, but even more as front marker, without it you can't calculate Rf-values which you need for molecular mass determination. > I would imagine I'll need to add B-ME to reduce the proteins before > running on the gel, but should I add the B-ME at the point I resuspend > the pellet, or only when I'm about to run an aliquot on a gel? Will I > need to add protease inhibitors, or will the Laemmli buffer itself take > care of that? Reducing agent is part of the 2x concentrate, usually the sample/buffer mixture is heated to get more efficient reduction. Many people do 2 min in a boiling water bath and for your purposes that is probably fine. With large proteins I found that 10 min at 60 degrees works better. Very few proteases work in hot SDS under reducing conditions, but there have been rare cases reported where that was a problem. The protease inhibitors should of course be present during the homogenisation of the cells and the centrifugation steps. Many (like PMSF) actually chemically inactivate proteases, solving the problem permanently anyway. From priyatripuraneni from gmail.com Wed Jun 18 11:02:31 2008 From: priyatripuraneni from gmail.com (priyadarsini Tripuraneni) Date: Wed Jun 18 11:36:50 2008 Subject: doubt regarding agrobacterium DNA restriction digestion Message-ID: hi all as i am attempting to digest agrobacterium DNA i am not able to do that. please help me regarding this. -- prasanthi From S.Lahham from med.umcg.nl Tue Jun 17 17:06:41 2008 From: S.Lahham from med.umcg.nl (S.Lahham@med.umcg.nl) Date: Wed Jun 18 11:36:57 2008 Subject: request for papers In-Reply-To: References: <715ec031-a920-4749-b514-429b695fb1fb@v26g2000prm.googlegroups.com> Message-ID: <20080618000641218.00000000316@129-125-166-139> Dear sir: Here is one -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Dr Engelbert Buxbaum Sent: dinsdag 17 juni 2008 21:31 To: methods@magpie.bio.indiana.edu Subject: Re: request for papers Am 12.06.2008, 04:53 Uhr, schrieb sheelu : > Dear all friends: > I have found two papers published in Nature & Science, the name > of which are "How biotech can transform biofuels".(Nature > biotechnology (2005 feb ) 26(2), 169-172; and another one is "Fuel > ethanol from cellulosic biomass" (Science 1991Mar 15:251(4999): > 1318-1323, I think > this paper would be great help to me.So anyone of here can help me to > get it? One method is to go to your library. If they do not have it, they can order a copy for you by interlibrary loan (not cheap though). The second method is to contact the authors and ask for a reprint. Their affiliation you get from the usual search tools, e.g. scholars.google.com or PubMed. Use email and you usually get an electronic copy within a few days. Exchange of reprints is a time-honored way of scholars to keep in contact. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From nick.theodorakis from gmail.com Wed Jun 18 10:57:27 2008 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Wed Jun 18 11:37:04 2008 Subject: E.coli strain recommendation, please References: Message-ID: <641384b3-ff96-4c1b-a104-6d343b539ab8@26g2000hsk.googlegroups.com> On Jun 18, 11:51?am, d...@no.email.thankstospam.net (DK) wrote: > Traditionally, for reasons I am not quite sure about, we use > Invitrogen's TOP10 cells for all routine cloning. There is one > serious problem with this strain, however. Cells grow too slowly. > E.g. to get a decent size clone, plates need to be incubated > 24 hours. In a typical overnight (4 pm - 9 am), clones are too > small. ?Also, "short" overnight liquid cultures are not saturated, > resulting in low plasmid yields. This is becoming a problem > when we want to work fast and not waste time waiting for the > bugs to grow. . > > So I am looking for alternatives that offer: > > 1. High transformation efficiency (both chemical > and electroporation). > 2. Low level of recombination. > 3. High plasmid yields and quality of DNA minipreps. > 4. Fast growth. > I like DH5alpha for my routine subcloning and plasmid prep needs. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From akhan357 from sbcglobal.net Wed Jun 18 14:17:08 2008 From: akhan357 from sbcglobal.net (AK) Date: Wed Jun 18 17:12:36 2008 Subject: request for papers References: <715ec031-a920-4749-b514-429b695fb1fb@v26g2000prm.googlegroups.com> Message-ID: initial poster has received both papers. thanks AK wrote in message news:mailman.484.1213807034.3533.methods@net.bio.net... Dear sir: Here is one -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Dr Engelbert Buxbaum Sent: dinsdag 17 juni 2008 21:31 To: methods@magpie.bio.indiana.edu Subject: Re: request for papers Am 12.06.2008, 04:53 Uhr, schrieb sheelu : > Dear all friends: > I have found two papers published in Nature & Science, the name > of which are "How biotech can transform biofuels".(Nature > biotechnology (2005 feb ) 26(2), 169-172; and another one is "Fuel > ethanol from cellulosic biomass" (Science 1991Mar 15:251(4999): > 1318-1323, I think > this paper would be great help to me.So anyone of here can help me to > get it? One method is to go to your library. If they do not have it, they can order a copy for you by interlibrary loan (not cheap though). The second method is to contact the authors and ask for a reprint. Their affiliation you get from the usual search tools, e.g. scholars.google.com or PubMed. Use email and you usually get an electronic copy within a few days. Exchange of reprints is a time-honored way of scholars to keep in contact. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From akhan357 from sbcglobal.net Wed Jun 18 14:30:27 2008 From: akhan357 from sbcglobal.net (AK) Date: Wed Jun 18 17:12:44 2008 Subject: request for paper References: Message-ID: Please read Dr. Buxbaum's suggestion on your previous request. Although people are more than willing to help under desperate conditions, it is not quite proper to routinely request papers on this forum. thanks AK "sheelu" wrote in message news:aa5c5dc2-6997-448a-a0a0-489ab96e8617@s21g2000prm.googlegroups.com... Dear friends: I need one paper published in FEMS microbiology letters, entitled " Cloning and characterization of two cellulase genes from Lentinula edodes" by Lee, C. C., Wong, D. W., and Robertson, G. H. 2001, 205: 355–360. if anyone of here can help me to get it? Thanks in advance! my mail id is gauravg...@gmail.com From higginsb78 from gmail.com Wed Jun 18 11:45:41 2008 From: higginsb78 from gmail.com (Brian P Higgins) Date: Wed Jun 18 17:12:57 2008 Subject: E.coli strain recommendation, please In-Reply-To: References: Message-ID: I'll second DH5alpha--works very well. I'd also suggest looking at your NEB catalog--they have several E. coli strains available for free (you pay shipping if you're not ordering something else). I know they offer several of the JM10X's, which are very handy (especially the recA strain, which is what you'd want). There are too many alternatives to list, so you'll have to be a bit more specific about the desired genotype for me to give more recommendations. I am not sure why you're having the problem with Top10. I've used it many times with great success and I have not found it to grow slowly at all. Could it be the level of antibiotic you are using? On Wed, Jun 18, 2008 at 11:51 AM, DK wrote: > Traditionally, for reasons I am not quite sure about, we use > Invitrogen's TOP10 cells for all routine cloning. There is one > serious problem with this strain, however. Cells grow too slowly. > E.g. to get a decent size clone, plates need to be incubated > 24 hours. In a typical overnight (4 pm - 9 am), clones are too > small. Also, "short" overnight liquid cultures are not saturated, > resulting in low plasmid yields. This is becoming a problem > when we want to work fast and not waste time waiting for the > bugs to grow. . > > So I am looking for alternatives that offer: > > 1. High transformation efficiency (both chemical > and electroporation). > 2. Low level of recombination. > 3. High plasmid yields and quality of DNA minipreps. > 4. Fast growth. > > TOP10 fit requirements #1-2, #3 if we wait long enough > but definitely not #4. > > Thanks, > > Dima > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From holeung from berkeley.edu Wed Jun 18 12:04:14 2008 From: holeung from berkeley.edu (Ho-Leung Ng) Date: Wed Jun 18 17:13:03 2008 Subject: E.coli strain recommendation, please Message-ID: <6678762a0806181004q1b5c8731i1bab9f89ff6ff295@mail.gmail.com> We also use DH5alpha for almost all cloning. It does not grow as quickly as I would like, but it is faster than what you describe for TOP10. Some companies sell fast growth cell lines for cloning, like NEB Turbo. ho From mlsulliv from wisc.edu Wed Jun 18 12:16:25 2008 From: mlsulliv from wisc.edu (Michael Sullivan) Date: Wed Jun 18 17:13:09 2008 Subject: doubt regarding agrobacterium DNA restriction digestion In-Reply-To: References: Message-ID: <39E83117-D974-4EA3-81FA-0C68EEB384E5@wisc.edu> Are you trying to digest plasmid DNA from agro to make sure you have transformed a binary vector construct? Yield and quality of DNA from agro are usually much lower than you would get from E. coli, so sometimes it is difficult to directly analyze DNA isolated from agro. I have had good luck using Qiagen plasmid minipreps. Usually, I get DNA of good enough yield and quality to see restriction digestion products. In the past, using simple alkaline lysis type procedures, results were typically more variable- sometimes I could see digestion products, sometimes not. However, I found the DNA was always of sufficient quality to retransform into E. coli. This means that to analyze a plasmid from agro, you may need to transform the DNA back into E. coli, where prepping and doing a digest will be trivial. If results of the DNA analysis from E. coli look good, you know your plasmid in agro was OK. Mike On Jun 18, 2008, at 11:02 AM, priyadarsini Tripuraneni wrote: > hi all > as i am attempting to digest agrobacterium DNA i am not able to do > that. > please help me regarding this. > > -- > prasanthi > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods From mprigge from indiana.edu Wed Jun 18 14:29:50 2008 From: mprigge from indiana.edu (Michael J. Prigge) Date: Wed Jun 18 17:13:14 2008 Subject: doubt regarding agrobacterium DNA restriction digestion Message-ID: What are the symptoms? Are you trying to digest plasmid DNA? If you can see the DNA on a gel but it looks uncut, it might be due to the differences methylation patterns. I don't know what sequences are methylated in Agro other than GANTC, but you should be able to find that info somewhere then check to see if your enzyme might be affected by that methylation. If you are not seeing anything on the gel, it is likely because binary vectors are (with a few excpetions) present at low copy numbers. I typically digest the plasmid yield from ~2 ml of culture for each lane on a gel. Mike > hi all > as i am attempting to digest agrobacterium DNA i am not able to do > that. > please help me regarding this. > > -- > prasanthi From L.Kamalian from liverpool.ac.uk Wed Jun 18 18:03:17 2008 From: L.Kamalian from liverpool.ac.uk (Kamalian, Laleh) Date: Thu Jun 19 11:37:29 2008 Subject: In vitro Assays for suspension cells Message-ID: <31DC2DFFD70C174B8E3072803F894F91046C9A@EVSSTAFF1.livad.liv.ac.uk> I am working on SCLC cell lines which grow in suspension. I have had lots of trouble so far as I am the only one in the lab working with this kind of cells. I would like to examine tumourogenisity, invasion and proliferation ability of my parental malignant cells comparing to the cells with reduced expression of specific genes. So far the only assay I have found to be straight forward is soft agar assay for tumourigenesis. Even that seems to be more of a proliferation assessment than adherence-independent grow, but at least it is doable. My main problem is with invasion assay because my cells wouldn't stay attached to the matrigel coated inserts. For proliferation, MTT and DMSO didn't give me a nice standard curve for different cell concentrations. Could any one please suggest any ideas? Thanks. Laleh From alain.tissier from librophyt.com Thu Jun 19 04:18:28 2008 From: alain.tissier from librophyt.com (Alain Tissier) Date: Thu Jun 19 11:37:37 2008 Subject: pGEM-T cloning Message-ID: <485A2464.4080805@librophyt.com> -- Alain TISSIER, PhD CEO LIBROPHYT Centre de Cadarache B?timent 185 13115 Saint-Paul-lez-Durance FRANCE www.librophyt.com Tel: +33 442 57 47 44 mob: +33 673 24 26 15 fax: +33 442 57 44 39 From clive.tregaskes from eu.effem.com Thu Jun 19 03:02:13 2008 From: clive.tregaskes from eu.effem.com (clive.tregaskes@eu.effem.com) Date: Thu Jun 19 11:39:22 2008 Subject: E.coli strain recommendation, please Message-ID: I've just started using TOP10 (came with one of the Invitrogen vectors so I thought I'd give it a go) and I've found variable colony size depending on the vector. One plasmid, pCDNA3.1, grows fine, nice big colonies, the second which is derived from pBluescript doesn't. These are barely visible after an overnight incubation. Haven't tried making plasmid from these- inserts amplify up quite nicely by PCR, but may give it a go just to see if they grow slowly in liquid culture as well Clive From pow.joshi from gmail.com Thu Jun 19 15:36:03 2008 From: pow.joshi from gmail.com (Pow Joshi) Date: Thu Jun 19 20:30:44 2008 Subject: pGEM-T cloning In-Reply-To: <485A2464.4080805@librophyt.com> References: <485A2464.4080805@librophyt.com> Message-ID: <710764ea0806191336s6efee876ycae08a333e16a43a@mail.gmail.com> Alain, nous ne avons pas re?u votre question.... pourriez-vous le renvoyer? merci Pow 2008/6/19 Alain Tissier : > > -- > Alain TISSIER, PhD > CEO > LIBROPHYT > Centre de Cadarache > B?timent 185 > 13115 Saint-Paul-lez-Durance > FRANCE > > www.librophyt.com > > Tel: +33 442 57 47 44 > mob: +33 673 24 26 15 > fax: +33 442 57 44 39 > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From jleonard.iii from gmail.com Thu Jun 19 16:50:44 2008 From: jleonard.iii from gmail.com (John Leonard) Date: Thu Jun 19 20:30:51 2008 Subject: Western Stripping Buffer? Message-ID: <83d01d5d0806191450x4d56e7e8n92f1302924d210c8@mail.gmail.com> Does anyone have a favorite buffer they have used to strip western blots for re-probing? I've found a number of recipes online, but I don't know which to choose. If you know of one that you particularly enjoy, please share! Thanks, John From jleonard.iii from gmail.com Thu Jun 19 17:26:03 2008 From: jleonard.iii from gmail.com (John Leonard) Date: Thu Jun 19 20:31:02 2008 Subject: Antibody Affinity Purification Message-ID: <83d01d5d0806191526x66391af4q1bb0d6a63c40007e@mail.gmail.com> Hello all, I have serum containing a custom polyclonal antibody and I'd like to affinity purify it via interaction with its ligand. I have His-tagged antigen that I would like to immobilize on a Ni-NTA column. I would, then, run diluted serum over this column to pull out any antibodies specific to my antigen, then selectively elute the antibody (ex. with high salt). I know this is possible and have an idea for a protocol, but I was wondering if any of you have done this before and have any pointers or protocols that have worked well for you (i.e. specific buffers, amounts of antibody and antigen to use, etc). Any suggestions are welcome! Thanks! John From L.Kamalian from liverpool.ac.uk Fri Jun 20 09:13:38 2008 From: L.Kamalian from liverpool.ac.uk (Kamalian, Laleh) Date: Fri Jun 20 12:27:15 2008 Subject: Western Stripping Buffer? References: <83d01d5d0806191450x4d56e7e8n92f1302924d210c8@mail.gmail.com> Message-ID: <31DC2DFFD70C174B8E3072803F894F91046C9C@EVSSTAFF1.livad.liv.ac.uk> I have used the following composition for stripping buffer. It has worked for me. To make 500 ml of Stripping Buffer: 62.5 mM Tris (3.8 grams of MW 121.1) 100 mM 2-mecaptoethanol ( 3.5 ml) 2% SDS (10 grams) PH to 6.7 Volume to 500 ml with distilled water. I usually incubate my membrane for 1/2 hour at 50'C on a shaker plate. Hope it helps. Laleh ________________________________ From: methods-bounces@oat.bio.indiana.edu on behalf of John Leonard Sent: Thu 19/06/2008 22:50 To: methods@magpie.bio.indiana.edu Subject: Western Stripping Buffer? Does anyone have a favorite buffer they have used to strip western blots for re-probing? I've found a number of recipes online, but I don't know which to choose. If you know of one that you particularly enjoy, please share! Thanks, John _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From virashkgupta from gmail.com Sat Jun 21 09:30:00 2008 From: virashkgupta from gmail.com (Virash Gupta) Date: Sat Jun 21 11:12:27 2008 Subject: Methods Digest, Vol 37, Issue 14 In-Reply-To: <200806181637.m5IGblO10574@net.bio.net> References: <200806181637.m5IGblO10574@net.bio.net> Message-ID: E.coli DH5-alha and sure are the best. High transformation efficiency is practically obtained when after developing competency cells are kept cold for atleast 24 hrs, and the stored cells are not older than 3 months. Plasmid copy number is decided both by the type of plasmid vector and the size of insert DNA ( insert genes with proteinase activity generally inhibit growth of transformed cells and need to be grown in th epresence of a suitable proteinase inhibitor. The minimum period for growth of E.coli clone from a single cell is 16 hrs- generally if medium pH is near 7 a conspicuous clone of good size is obtained in 20 hrs at 37C, although it will be visible within 14-16 hrs. 4PM- 9AM period is not as suitable practically as if you get good sized clone at around 9 or 10 AM, you will not have enough time on same day to get good groth in LB for plasmid isolation. So let it grow till 3 PM, inoculate in 3ml LB let grow overnight before you isolate MP plasmid next day in the morning and analyze the same till evening. For good yield of plasmid- while using alkaline lysis method- never use old P1, P2, P3 solutions that are stored at RT for more than one month. It is more true for Lysis solution which due to presence of NAOH absorbs CO2 from atmosphere that results in decreased alkaline pH to reduce lysis activity of this solution. Make solutions in bulk, store the stock solutions at low temperature in tightened reagent bottles- draw small working usable volumes in separate tubes for regular use. It will solve many problems. Low plasmid yields also result from improper handling of procedure which results in residual endonuclease activities due to contaminating (precipitated) proteins. On 6/18/08, methods-request@oat.bio.indiana.edu wrote: > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. Re: Poor resolution when isolating ssDNA? (cheetah.zao@gmail.com) > 2. Advice needed for Western blotting (Peter Ellis) > 3. T4 PNK (peter) > 4. Re: request for papers (Dr Engelbert Buxbaum) > 5. Re: Advice needed for Western blotting (Dr Engelbert Buxbaum) > 6. E.coli strain recommendation, please (DK) > 7. doubt regarding agrobacterium DNA restriction digestion > (priyadarsini Tripuraneni) > 8. Re: E.coli strain recommendation, please (Nick Theodorakis) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Mon, 16 Jun 2008 23:12:20 -0700 (PDT) > From: "cheetah.zao@gmail.com" > Subject: Re: Poor resolution when isolating ssDNA? > To: methods@net.bio.net > Message-ID: > <5145efed-bf2d-45bd-be7d-f91b3dbca297@w1g2000prd.googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > 70% enthenol precipitation in the presence of 50 mM MgCl2 may work. > > > > On Jun 13, 5:56 am, Evan Reed wrote: > > Hej, > > > > I am trying to isolate ssDNA after binding dsDNA oligos with > > biotinylated antisense strands to strep microbeads, denaturing with > > NaOH, then adding the supernatant containing the sense ssDNA to a > > NAP-5 sephadex desalting column. Unfortunately, I am getting terrible > > resolution when I try to elute on the column -- even after 2 mL of > > elution, I am still getting detectable levels of ssDNA in the eluate > > (dsDNA oligos of the same length elute just fine in 0.5 mL, with > > acceptable resolution). The amount of DNA loaded into the column > > should be well below the binding capacity so I am not sure why this is > > happening. Has anyone done this before? Is there a better way to > > purify the ssDNA instead of this column? > > > > Thanks, > > > > ER > > > > ------------------------------ > > Message: 2 > Date: Tue, 17 Jun 2008 12:06:12 +0100 > From: Peter Ellis > Subject: Advice needed for Western blotting > To: methods@net.bio.net > Message-ID: > Content-Type: TEXT/PLAIN; charset=US-ASCII; format=flowed > > Hiya, > > I am just starting up a project investigating two novel proteins in > testis function. My background is in molecular genetics and transcription > profiling, so protein work is all pretty new to me. > > We have peptide antibodies prepared (or in the works) for both proteins - > the first step is obviously to characterise the antibodies. We're using > peptide antibodies rather than antibodies to recombinant protein because > there are many related genes, and we want to avoid cross-reactivity. > > We have ORFs cloned for each of the genes and their close relatives. The > aim is to use in vitro transcription/translation to generate pure > proteins in order to check the antibody specificity. Once we know > that the antibodies do actually detect our proteins of interest (and not > the relatives), we can move on to other more interesting questions! > > So the first plan is Western blots using whole testis extracts to see > whether we have a band of the expected size in testis. We'll then do > further Western blots of testis extract alongside the various in vitro > translated proteins in order to check AB specificity. At this point I'm > looking for advice / sanity checking on how to extract my proteins and > what sort of gels to use for the Western blots. > > On looking around in the literature, I'm finding myself a little > bewildered by buffer compositions - many papers refer to "standard RIPA > buffer" or "standard Laemmli sample buffer", and yet there seem to be as > many different recipes as there are laboratories. Secondly, it looks like > the most common buffers simply won't work for my proteins! > > Details below for each of the proteins of interest: > > ************************************************* > > Protein 1: H2AL1 > ---------------- > > This one is a novel histone. I've looked at the QIAGEN QProteome Nuclear > kit, but I'm loath to commit to using that as it doesn't tell you what any > of the buffers are. I can't even tell if it's an acid extraction or a > salt extraction! Also, it's very expensive for the amount of tissue you > can process with it. > > So I'd prefer to do my own histone extraction if possible. I believe that > means I want to prepare purified nuclei and then do an acid extraction. > Previously I've done nuclear preps when preparing high molecular weight > gDNA - will the same buffers be appropriate for protein work? Protocol is > as follows: > > * Homogenise tissue at low speed in lysis buffer [0.05M TrisCl pH 7.5, 1mM > EDTA, 5mM MgCl2, 50mM NaCl, 5% glycerol, 1% Triton X-100, 1% B-ME], using > 100 mg tissue / ml buffer. > > * Centrifuge to spin down nuclei > > * Wash in lysis buffer and spin down again > > > Once I've got the purified nuclei, the histone prep looks admirably > straightforward. According to Abcam, you just resuspend the nuclear > pellet in 0.2M HCl and leave it overnight at 4 degrees C. Centrifuge to > spin down debris and keep the supernatant. However, other protocols seem > to use different acid concentrations, or H2SO4 instead of HCl, and may > even include B-ME in the histone extraction buffer. > > One protocol I Googled called for you to neutralise the preparation with > KOH afterwards - is that really called for? Wouldn't it just drop the > proteins straight back out of solution again? > > Once I've got my histone prep, what buffer should I use for running them > on an SDS-PAGE gel? Standard Laemmli sample buffer? If so, *which* > standard Laemmli buffer? Will a normal SDS-PAGE gel work OK, or will > there be pH issues due to the sample being in pretty much neat acid? > Generally the lab uses Novex gels in a XCell mini-blot module. For a > histone (size ~17 kDa), it'll be quite a high percentage gel. > > > ************************************************* > > > Protein 1: mgclh > ------------------ > > Localisation of this protein is unknown, but a closely related gene > (Gmcl1) is known to be a nuclear lamina protein. Gmcl1 is RIPA-insoluble, > so it's likely mgclh will also be RIPA-insoluble. Protein size is > expected to be around 55 kDa. > > The plan here is to do a standard protein extraction by homogenising > testis tissue in RIPA + protease inhibitors. Retain the supernatant (to > get the RIPA-soluble fraction), and then dissolve the pellet in something > (to get the RIPA-insoluble fraction). Question is what to dissolve the > pellet in. > > The original literature on Gmcl1 said they resuspended the RIPA-insoluble > pellet in "SDS-polyacrylamide gel electrophoresis sample buffer", but > doesn't give the composition. I would assume this is 1x Laemmli sample > buffer. Does this sound plausible? If so, what recipe for Laemmli buffer > would you recommend? I've found half a dozen different recipes, and the > only common factor between them is ~60-100mM Tris pH 6.8 and 2% SDS. > There are variants with and without DTT, with and without B-ME, with > and without loading dye, using sucrose or glycerol for increased density! > > I would imagine I'll need to add B-ME to reduce the proteins before > running on the gel, but should I add the B-ME at the point I resuspend the > pellet, or only when I'm about to run an aliquot on a gel? Will I need to > add protease inhibitors, or will the Laemmli buffer itself take care of > that? > > ************************************************* > > Thank you for your time and patience - sorry for asking so many questions, > most of which will undoubtedly have obvious answers. Probably several > mutually incompatible obvious answers, mind you, but that's biology for > you. My theory is that it's better to ask silly questions and look daft > than to forge ahead without asking and cock something up! > > Thanks, > Peter Ellis > > > ------------------------------ > > Message: 3 > Date: Tue, 17 Jun 2008 11:19:15 -0700 (PDT) > From: peter > Subject: T4 PNK > To: methods@net.bio.net > Message-ID: > <28b39b3f-6f52-4c5e-baa3-37a22181d61c@8g2000hse.googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Hi all, > > have kinda silly question: Can T4PNK use dATP instead of rATP to > phosphorilate DNA? > Thanks , > Peter > > > ------------------------------ > > Message: 4 > Date: Tue, 17 Jun 2008 15:30:32 -0400 > From: "Dr Engelbert Buxbaum" > Subject: Re: request for papers > To: methods@net.bio.net > Message-ID: > Content-Type: text/plain; format=flowed; delsp=yes; > charset=iso-8859-15 > > Am 12.06.2008, 04:53 Uhr, schrieb sheelu : > > > Dear all friends: > > I have found two papers published in Nature & Science, the name > > of which > > are "How biotech can transform biofuels".(Nature biotechnology (2005 > > feb ) 26(2), 169-172; and another one is > > "Fuel ethanol from cellulosic biomass" (Science 1991Mar 15:251(4999): > > 1318-1323, I think > > this paper would be great help to me.So anyone of here can help me to > > get it? > > One method is to go to your library. If they do not have it, they can > order a copy for you by interlibrary loan (not cheap though). > > The second method is to contact the authors and ask for a reprint. Their > affiliation you get from the usual search tools, e.g. scholars.google.com > or PubMed. Use email and you usually get an electronic copy within a few > days. Exchange of reprints is a time-honored way of scholars to keep in > contact. > > > ------------------------------ > > Message: 5 > Date: Tue, 17 Jun 2008 17:41:45 -0400 > From: "Dr Engelbert Buxbaum" > Subject: Re: Advice needed for Western blotting > To: methods@net.bio.net > Message-ID: > Content-Type: text/plain; format=flowed; delsp=yes; > charset=iso-8859-15 > > Am 17.06.2008, 07:06 Uhr, schrieb Peter Ellis : > > > We have peptide antibodies prepared (or in the works) for both proteins > > - the first step is obviously to characterise the antibodies. We're > > using peptide antibodies rather than antibodies to recombinant protein > > because there are many related genes, and we want to avoid > > cross-reactivity. > > That is fine, just remember that the epitope may be hidden in native > proteins, thus the AB may not work e.g. in immunoprecipitation. In Western > you deal with denatured proteins, so that is not a problem. > > > We have ORFs cloned for each of the genes and their close relatives. > > The aim is to use in vitro transcription/translation to generate pure > > proteins in order to check the antibody specificity. Once we know that > > the antibodies do actually detect our proteins of interest (and not the > > relatives), we can move on to other more interesting questions! > > In addition to testing against individual proteins you should test against > a crude tissue lysate. You should see only a single band. Even more > telling would be a blot from a 2D-gel (SDS-PAGE after IEF). > > > At this point I'm looking for advice / sanity checking on how to > > extract my proteins and what sort of gels to use for the Western blots. > > The most common method is SDS-PAGE according to Laemmli (Laemmli: Nature > 227 (1970) 680 and Laemmli & Favre, J. Mol. Biol. 80 (1973) 575-599). > Separation is by molecular mass. For more specific detection, this is > preceeded by isoelectric focussing, a method that separates by isoelectric > point. See J. Klose and U. Kobalz: Electrophoresis 16 (1995) 1034-1059 > > > On looking around in the literature, I'm finding myself a little > > bewildered by buffer compositions - many papers refer to "standard RIPA > > buffer" or "standard Laemmli sample buffer", and yet there seem to be as > > many different recipes as there are laboratories. Secondly, it looks > > like the most common buffers simply won't work for my proteins! > > It's not as bad as that. The Laemmli buffer is a fairly good bet. > > > This one is a novel histone. I've looked at the QIAGEN QProteome > > Nuclear kit, but I'm loath to commit to using that as it doesn't tell > > you what any of the buffers are. I can't even tell if it's an acid > > extraction or a salt extraction! Also, it's very expensive for the > > amount of tissue you can process with it. > > Histones have a high positive charge, to get a reasonable estimate of > their molecular mass from PAGE, use the positive detergent CTAB rather > than the negatively charged SDS (E. Buxbaum: Anal. Biochem. 314 {2003} > 70-76) > > > So I'd prefer to do my own histone extraction if possible. I believe > > that means I want to prepare purified nuclei and then do an acid > > extraction. Previously I've done nuclear preps when preparing high > > molecular weight gDNA - will the same buffers be appropriate for protein > > work? Protocol is as follows: > > > > Homogenise tissue at low speed in lysis buffer [0.05M TrisCl pH 7.5, 1mM > > EDTA, 5mM MgCl2, 50mM NaCl, 5% glycerol, 1% Triton X-100, 1% B-ME], > > using 100 mg tissue / ml buffer. > > I'd probably use KCl rather than NaCl, as the cell interior is high in K > and low in Na. I'd also reduce [Mg] to 1 mM or so to discourage > metal-dependent proteases. The Triton I'd leave out at this step and > reduce bME to 1 mM (or replace it with DTT, which has a more appropriate > redox potential). You'll need a protease inhibitor coctail (EDTA, PMSF, > Pepstatin, Leupeptin, e-aminocaproic acid). > > > Once I've got the purified nuclei, the histone prep looks admirably > > straightforward. According to Abcam, you just resuspend the nuclear > > pellet in 0.2M HCl and leave it overnight at 4 degrees C. Centrifuge to > > spin down debris and keep the supernatant. However, other protocols > > seem to use different acid concentrations, or H2SO4 instead of HCl, and > > may even include B-ME in the histone extraction buffer. > > The acid is quite irrelevant, the idea is simply to increase the > solubility of the highly charged histons and to pellet as many of the > other proteins as possible. The bME protects cysteine -SH groups from > oxidation by air. However, this extraction procedure is too selective for > your purposes, you will not know whether there are cross-reacting proteins > in the pellet. > > > > One protocol I Googled called for you to neutralise the preparation with > > KOH afterwards - is that really called for? Wouldn't it just drop the > > proteins straight back out of solution again? > > No. But you need a pH near neutral and as low an ion concentration as > possible for the electrophoresis step. Therefore solubilisation in sample > buffer rather than HCl is better. That also gives you an idea about the > cross-reactions of your antibody. > > > For a histone (size ~17 kDa), it'll be quite a high percentage gel. > > I find gradient gels (5-15 or 20%) most convenient > > > > > Protein 1: mgclh > > ------------------ > > > > RIPA-insoluble, so it's likely mgclh will also be RIPA-insoluble. > > Protein size is expected to be around 55 kDa. > > Again the use of CTAB electrophoresis may help, as CTAB can solubilise > proteins very efficiently. > > > The original literature on Gmcl1 said they resuspended the > > RIPA-insoluble pellet in "SDS-polyacrylamide gel electrophoresis sample > > buffer", but doesn't give the composition. I would assume this is 1x > > Laemmli sample buffer. Does this sound plausible? If so, what recipe > > for Laemmli buffer would you recommend? I've found half a dozen > > different recipes, and the only common factor between them is ~60-100mM > > Tris pH 6.8 and 2% SDS. There are variants with and without DTT, with > > and without B-ME, with and without loading dye, using sucrose or > > glycerol for increased density! > > The buffer is actually double concentrated, once you mix it with an equal > volume of sample you get the working strenght. DTT and bME reduce > disulphide bonds and thus lead to more complete unfolding of the proteins. > DTT (Clelands reagent) smells less and has a better standard redox > potential for this purpose. However, it is more expensive than bME. Wether > you use glycerol or sucrose to increase density really doesn't matter, you > just need either for proper gel loading, its physics, not chemistry. The > dye is important not only to aid in loading, but even more as front > marker, without it you can't calculate Rf-values which you need for > molecular mass determination. > > > I would imagine I'll need to add B-ME to reduce the proteins before > > running on the gel, but should I add the B-ME at the point I resuspend > > the pellet, or only when I'm about to run an aliquot on a gel? Will I > > need to add protease inhibitors, or will the Laemmli buffer itself take > > care of that? > > Reducing agent is part of the 2x concentrate, usually the sample/buffer > mixture is heated to get more efficient reduction. Many people do 2 min in > a boiling water bath and for your purposes that is probably fine. With > large proteins I found that 10 min at 60 degrees works better. Very few > proteases work in hot SDS under reducing conditions, but there have been > rare cases reported where that was a problem. The protease inhibitors > should of course be present during the homogenisation of the cells and the > centrifugation steps. Many (like PMSF) actually chemically inactivate > proteases, solving the problem permanently anyway. > > > ------------------------------ > > Message: 6 > Date: Wed, 18 Jun 2008 15:51:44 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: E.coli strain recommendation, please > To: methods@net.bio.net > Message-ID: > > Traditionally, for reasons I am not quite sure about, we use > Invitrogen's TOP10 cells for all routine cloning. There is one > serious problem with this strain, however. Cells grow too slowly. > E.g. to get a decent size clone, plates need to be incubated > 24 hours. In a typical overnight (4 pm - 9 am), clones are too > small. Also, "short" overnight liquid cultures are not saturated, > resulting in low plasmid yields. This is becoming a problem > when we want to work fast and not waste time waiting for the > bugs to grow. . > > So I am looking for alternatives that offer: > > 1. High transformation efficiency (both chemical > and electroporation). > 2. Low level of recombination. > 3. High plasmid yields and quality of DNA minipreps. > 4. Fast growth. > > TOP10 fit requirements #1-2, #3 if we wait long enough > but definitely not #4. > > Thanks, > > Dima > > > > ------------