From hzhen from freeuk.com Thu Nov 1 00:01:22 2007 From: hzhen from freeuk.com (ChenHA) Date: Thu Nov 1 08:27:46 2007 Subject: why EDTA is used for lysozyme action In-Reply-To: References: Message-ID: <1193893481.31711.0@proxy01.news.clara.net> chiranjit chowdhury wrote: > Dear friends, I got protocol from pharmacia for Gram negative bacterial cell > lysis with lysozyme and EDTA. I have gone through several literature where > they have used EDTA with lysozyme mediated cell lysis of Gram negative > bacteria but not in case of Gram positive bacteria. > Can anybody tell me why EDTA is used specially for Gram negative bacteria, > the exact mechanism behind it > Regards. > > EDTA chelates divalent cations like calcium and magnesium. IIRC, these divalent cations are important for maintaining the structures on the cell surface. Removing them destablises these cell surface structures and makes it easier to lyse the cells. As far as I can remember, having too much as well as too little calcium and magnesium can change the cell surface structures which, if I am remember correctly, is I think one reason why you use calcium when you prepare competent cells to make the cells more permeable to DNA (calcium also help screen out the charges allowing the DNA to stick to the cell surface). From sarovarbhee from yahoo.co.in Thu Nov 1 00:45:03 2007 From: sarovarbhee from yahoo.co.in (bheemathati sarovar) Date: Thu Nov 1 08:35:52 2007 Subject: RT-PCR Troubleshooting - HELP! Message-ID: <961839.87061.qm@web7607.mail.in.yahoo.com> hi, Iam Miss.B.Sarovar,research scholar in the virology department working on ssRNA plant viruses.I would like to explain my problem as:i collected the sample,done rna extraction and pcr too.Initially i got positive results for the above.But later when i want to optomize the expt or to do the same iam not getting the same.for this i checked my work book and analysed in many ways.when iam doing the expt for the second time iam getting streak in tne specified region.hope i may get the answer at the earliest. thanking you sarovar Chat on a cool, new interface. No download required. Go to http://in.messenger.yahoo.com/webmessengerpromo.php From sudhee26 from gmail.com Thu Nov 1 08:42:17 2007 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu Nov 1 09:26:04 2007 Subject: why EDTA is used for lysozyme action In-Reply-To: <1193893481.31711.0@proxy01.news.clara.net> References: <1193893481.31711.0@proxy01.news.clara.net> Message-ID: I doubt whether it has to do with the cell wall..probably lipoppolysaccharide and protein outermembrane is succeptible to EDTA disruption, than the peptidoglycan layer of gram positive bacteria. Correct me. Sudheendra NBRC On 11/1/07, ChenHA wrote: > > chiranjit chowdhury wrote: > > Dear friends, I got protocol from pharmacia for Gram negative bacterial > cell > > lysis with lysozyme and EDTA. I have gone through several literature > where > > they have used EDTA with lysozyme mediated cell lysis of Gram negative > > bacteria but not in case of Gram positive bacteria. > > Can anybody tell me why EDTA is used specially for Gram negative > bacteria, > > the exact mechanism behind it > > Regards. > > > > > > EDTA chelates divalent cations like calcium and magnesium. IIRC, these > divalent cations are important for maintaining the structures on the > cell surface. Removing them destablises these cell surface structures > and makes it easier to lyse the cells. > > As far as I can remember, having too much as well as too little calcium > and magnesium can change the cell surface structures which, if I am > remember correctly, is I think one reason why you use calcium when you > prepare competent cells to make the cells more permeable to DNA (calcium > also help screen out the charges allowing the DNA to stick to the cell > surface). > _______________________________________________ > 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 hzhen from freeuk.com Thu Nov 1 09:45:44 2007 From: hzhen from freeuk.com (ChenHA) Date: Thu Nov 1 22:07:46 2007 Subject: why EDTA is used for lysozyme action In-Reply-To: References: <1193893481.31711.0@proxy01.news.clara.net> Message-ID: <1193928543.8488.0@proxy02.news.clara.net> Sudheendra Rao N R wrote: > I doubt whether it has to do with the cell wall..probably > lipoppolysaccharide and protein outermembrane is succeptible to EDTA > disruption, than the peptidoglycan layer of gram positive bacteria. > > Correct me. > Who said anything about cell wall? From ivanoov from gmail.com Thu Nov 1 10:17:16 2007 From: ivanoov from gmail.com (chovek69) Date: Thu Nov 1 22:07:52 2007 Subject: inhibitet PCR reaction In-Reply-To: References: Message-ID: <1193930236.767219.75180@50g2000hsm.googlegroups.com> On Oct 31, 5:48 pm, "Omar Hamarsheh" wrote: > Dear Colleges, > > I am working with ITS1 PCR using Leishmania DNA samples extracted from > filter papers, the samples where originated from skin scraping and > contain blood. I used inhibitor control for each sample . few samples > were worked and gave positive. most of the others show no PCR bands, > the inhibition control sho also no band which mean that there is an > inhibition of the PCR reaction probably caused from something in > blood. > I would like to ask colleges who are working with clinical samples if > they have an inhibitted PCR reaction what could be done in this > regard. > > thanks alot in advance for any help or suggestion. > > Omar Hamarsheh > > Charite school of Medicine > Dorotheenstr. 96 > 10117 Berlin > Germany Hi I have quite a bit experience with blood inhibited PCRs. The main inhibitor is the hem from the hemoglobine but there are others. The best remedy so far is the addition of nonacetylated Bovine Serum Albumine (BSA) from Sigma, in 0.1 to 0.8 µg/µL final concentration. Put in the PCR mix and sing. It depends also from the Taq but in general all works the same. There are other polymerases that are not inhibeted from the blood but they are expensive. (see for example this http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=87622 Hope this helps Regards From R.Jayakumar from roswellpark.org Thu Nov 1 10:42:10 2007 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Thu Nov 1 22:08:01 2007 Subject: RT-PCR Troubleshooting - HELP! In-Reply-To: <961839.87061.qm@web7607.mail.in.yahoo.com> Message-ID: <97101976F8A044468CA74FE11883B90E173E7917@VISTA.roswellpark.org> Is the streak you are specifying the PCR amplicon?? If so, did you do an actin or GAPDH or 18S control PCR on the samples. How did they work out? Did you try buying new primers and using that? Let me know. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of bheemathati sarovar Sent: Thursday, November 01, 2007 1:45 AM To: methods@magpie.bio.indiana.edu Subject: RT-PCR Troubleshooting - HELP! hi, Iam Miss.B.Sarovar,research scholar in the virology department working on ssRNA plant viruses.I would like to explain my problem as:i collected the sample,done rna extraction and pcr too.Initially i got positive results for the above.But later when i want to optomize the expt or to do the same iam not getting the same.for this i checked my work book and analysed in many ways.when iam doing the expt for the second time iam getting streak in tne specified region.hope i may get the answer at the earliest. thanking you sarovar Chat on a cool, new interface. No download required. Go to http://in.messenger.yahoo.com/webmessengerpromo.php _______________________________________________ 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 rcker_ros from yahoo.co.in Sat Nov 3 01:07:57 2007 From: rcker_ros from yahoo.co.in (rosmy sebastian) Date: Sat Nov 3 06:49:33 2007 Subject: purification of protein s from gel -reg Message-ID: <222615.3076.qm@web94411.mail.in2.yahoo.com> Respected sir , I saw a link about a protocol for purifying proteins from SDS-gel. Could you please send the same to the above e-mail? Thanking you, regards Ros --------------------------------- Flying to Bangalore or Bhopal? Search for tickets here. From sbeug from ohri.ca Tue Nov 6 14:06:58 2007 From: sbeug from ohri.ca (Beug, Shawn) Date: Tue Nov 6 23:26:02 2007 Subject: Antibody binding to nonpermeabilized cells Message-ID: <7C08583D5DA01D4D963926046238C6C708250C@ohexch05.civic1.ottawahospital.on.ca> Hi all, I am interested in calculating the ratio of intracellular and extracellular GFP-tagged protein in primary neurons. I am trying to demonstrate that perturbation of posttranslational modification affects extracellular/intracellular sublocalization. I was wondering if immunnostaining for transfected neurons with an anti-GFP antibody (using a Cy3-conjugated secondary antibody) would be the best way to calculate this ratio. I have done this before in the past but am unsure if antibodies would stain intracellular proteins, and as a result, give me the wrong numbers. Cheers, Shawn -------------------- 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 ebs15242 from creighton.edu Tue Nov 6 15:10:43 2007 From: ebs15242 from creighton.edu (Ed Siefker) Date: Tue Nov 6 23:26:12 2007 Subject: Acid Phenol/chloroform Message-ID: <4730CA43.90307@creighton.edu> I got a kit from Ambion to do some RACE, this kit requires acid phenol/chloroform but doesn't tell me what the composition of this is. I called Ambion and they didn't know. I couldn't find a recipe in Maniatis either. I have a brand new bottle of phenol ph 6.7. What do I need to add to make "acid phenol/chloroform"? Thanks. -Ed From sudhee26 from gmail.com Wed Nov 7 00:47:32 2007 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Wed Nov 7 11:07:10 2007 Subject: Acid Phenol/chloroform In-Reply-To: <4730CA43.90307@creighton.edu> References: <4730CA43.90307@creighton.edu> Message-ID: Hi, I got this from ambion website :) i hope this will help you. http://www.ambion.com/techlib/tb/tb_158.html On Nov 7, 2007 1:40 AM, Ed Siefker wrote: > I got a kit from Ambion to do some RACE, this kit requires acid > phenol/chloroform but doesn't tell me what the composition of this is. > I called Ambion and they didn't know. I couldn't find a recipe in > Maniatis either. > > I have a brand new bottle of phenol ph 6.7. What do I need to add to > make "acid phenol/chloroform"? Thanks. > -Ed > > _______________________________________________ > 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 pow.joshi from gmail.com Wed Nov 7 10:48:50 2007 From: pow.joshi from gmail.com (Pow Joshi) Date: Wed Nov 7 11:07:16 2007 Subject: Acid Phenol/chloroform In-Reply-To: <4730CA43.90307@creighton.edu> References: <4730CA43.90307@creighton.edu> Message-ID: <710764ea0711070748h1a076811s258fd3b42dd212a2@mail.gmail.com> On 06/11/2007, Ed Siefker wrote: > > I got a kit from Ambion to do some RACE, this kit requires acid > phenol/chloroform but doesn't tell me what the composition of this is. > I called Ambion and they didn't know. I couldn't find a recipe in > Maniatis either. > > I have a brand new bottle of phenol ph 6.7. What do I need to add to > make "acid phenol/chloroform"? Thank You can get the recipie for making the same from either Sambrook etal., or > Current methods in molecular biology. Best Pow -Ed > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From sudhee26 from gmail.com Wed Nov 7 00:51:29 2007 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Wed Nov 7 11:07:22 2007 Subject: purification of protein s from gel -reg In-Reply-To: <222615.3076.qm@web94411.mail.in2.yahoo.com> References: <222615.3076.qm@web94411.mail.in2.yahoo.com> Message-ID: Hi, Check out this link. http://www.google.co.in/url?sa=t&ct=res&cd=1&url=http%3A%2F%2Fwww.piercenet.com%2Ffiles%2FTR0051dh4-Elute-from-polyacrylamide.pdf&ei=5lExR8HRE4GKtALZzehy&usg=AFQjCNGVWCucXxdkowOSbB3OcCjy8VvPEQ&sig2=4SM3TStbZJo7WzsvJLbDDg Sudheendra. On Nov 3, 2007 11:37 AM, rosmy sebastian wrote: > Respected sir , > I saw a link about a protocol for purifying proteins from SDS-gel. Could > you please send the same to the above e-mail? > Thanking you, > regards Ros > > > --------------------------------- > Flying to Bangalore or Bhopal? Search for tickets here. > _______________________________________________ > 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 ebs15242 from creighton.edu Wed Nov 7 12:05:49 2007 From: ebs15242 from creighton.edu (Ed Siefker) Date: Wed Nov 7 15:04:53 2007 Subject: Acid Phenol/chloroform In-Reply-To: <20071107125011.e0sffhsdes4kkocc@webmail.irb.hr> References: <4730CA43.90307@creighton.edu> <20071107125011.e0sffhsdes4kkocc@webmail.irb.hr> Message-ID: <4731F06D.5040503@creighton.edu> Ah, thanks a lot. Looks like what I have is already buffered to be neutral and it might take a lot of acid to get it back down to the right pH. So I'll just have to hold off and get the right stuff. -Ed Jasenka Pigac wrote: > Phenol is acid as such when melted. If you add either water or > chloroform it will remain acid. I suppose it means to melt phenol > (Sigma) and add the same volume of chloroform - you will get acid > phenol/chloroform. The one you have has been pH adjusted to be neutral > (pH 6.7). > >> I got a kit from Ambion to do some RACE, this kit requires acid >> phenol/chloroform but doesn't tell me what the composition of this is. >> I called Ambion and they didn't know. I couldn't find a recipe in >> Maniatis either. >> >> I have a brand new bottle of phenol ph 6.7. What do I need to add to >> make "acid phenol/chloroform"? Thanks. >> -Ed >> >> _______________________________________________ >> Methods mailing list >> Methods@net.bio.net >> http://www.bio.net/biomail/listinfo/methods > > From stewjw from gmail.com Wed Nov 7 12:09:16 2007 From: stewjw from gmail.com (StewJW) Date: Wed Nov 7 15:05:00 2007 Subject: Acid Phenol/chloroform In-Reply-To: References: <4730CA43.90307@creighton.edu> Message-ID: <1194455356.388408.136740@v3g2000hsg.googlegroups.com> Great Ambion technical support?! I am being ironic for American readers. From nick_theodorakis from hotmail.com Wed Nov 7 13:12:49 2007 From: nick_theodorakis from hotmail.com (Nick Theodorakis) Date: Wed Nov 7 15:05:06 2007 Subject: Acid Phenol/chloroform In-Reply-To: References: Message-ID: <1194459169.660890.101570@v3g2000hsg.googlegroups.com> On Nov 6, 3:10 pm, Ed Siefker wrote: > I got a kit from Ambion to do some RACE, this kit requires acid > phenol/chloroform but doesn't tell me what the composition of this is. > I called Ambion and they didn't know. I couldn't find a recipe in > Maniatis either. > > I have a brand new bottle of phenol ph 6.7. What do I need to add to > make "acid phenol/chloroform"? Water-saturated phenol is "acid phenol (phenol has a weakly ionizable hydrogen)." What you have is likely buffered. Start with solid phenol crystals, and saturate it with good water (you might need to do it a couple of times). Standard safety disclaimers apply (wear gloves and eye and body protection, etc.). Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From nick_theodorakis from hotmail.com Wed Nov 7 13:22:38 2007 From: nick_theodorakis from hotmail.com (Nick Theodorakis) Date: Wed Nov 7 15:05:11 2007 Subject: why EDTA is used for lysozyme action In-Reply-To: References: <1193893481.31711.0@proxy01.news.clara.net> Message-ID: <1194459758.417703.48070@k79g2000hse.googlegroups.com> On Nov 1, 8:42 am, "Sudheendra Rao N R" wrote: > I doubt whether it has to do with the cell wall..probably > lipoppolysaccharide and protein outermembrane is succeptible to EDTA > disruption, than the peptidoglycan layer of gram positive bacteria. > > Correct me. That's pretty close. The substrate for lysozyme is peptidoglycan, which is the major component of the cell wall in gram-positive bacteria. Gram negs also have a (thin) peptidoglycan layer, but it is surrounded by an outer membrane that has LPS in it. Without additional treatment, this outer membrane protects gram neg bacteria from lysozyme. EDTA can disrupt this layer and allow lysozyme to have access to the peptidoglycan. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From ben.long from yourfinger.anu.edu.au Wed Nov 7 22:05:15 2007 From: ben.long from yourfinger.anu.edu.au (Bean Long) Date: Thu Nov 8 12:28:56 2007 Subject: Incomplete translation of N-term His-tagged protein in E.coli Message-ID: <47327ceb$1@clarion.carno.net.au> Hi all, I am expressing a protein in E. coli which has a cleavable N-terminal His-tag. The purified IMAC elaute prior to tag cleavage contains the tagged protein plus a large number of lower mass bands on SDS-PAGE. These masses all drop accordingly in size when the tag is cleaved, suggesting they are incomplete translation products (i.e. N-terminus is intact but C-terminus is shortened) of the protein I'm after. Can anyone offer any suggestion about, say, growth conditions or other other treatments which might help avoid this? I am growing cultures overnight at 23 ?C with 0.5 mM IPTG induction and harvesting using pretty standard methods (spin, lyse in IMAC binding buffer, etc). The cleavage requires a specific protease, so a protease cocktail is not used, just PMSF. Nonetheless, I am going to try using a cocktail next time to see if this might be a C-terminal protease problem. I am also expressing a short form of the same protein (which has a portion of the N-terminus missing) which does not appear to have the same problem, but this may be due to relatively low yields resulting from an apparent dimerisation and inaccessibility to IMAC in the short form. Ideas would be welcome. Cheers, -- Bean Remove "yourfinger" before replying From hroychow from nmsu.edu Wed Nov 7 18:42:51 2007 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Thu Nov 8 12:29:03 2007 Subject: Acid Phenol/chloroform In-Reply-To: <4730CA43.90307@creighton.edu> References: <4730CA43.90307@creighton.edu> Message-ID: <1205.128.123.174.0.1194478971.squirrel@webmail.nmsu.edu> The pH of the acid phenol is usually kept around 4.0. We used to make it in lab with Tri.HCl, but Sigma sells the stuff for so cheap that it doesn't pay to even equilibrate your own. Sigma sells both Phenol and Phenol Chloroform with pH8.0 or pH4 buffers. > I got a kit from Ambion to do some RACE, this kit requires acid > phenol/chloroform but doesn't tell me what the composition of this is. > I called Ambion and they didn't know. I couldn't find a recipe in > Maniatis either. > > I have a brand new bottle of phenol ph 6.7. What do I need to add to > make "acid phenol/chloroform"? Thanks. > -Ed > > _______________________________________________ > 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 prae from gmx.net Thu Nov 8 04:07:51 2007 From: prae from gmx.net (Christian Praetorius) Date: Thu Nov 8 12:29:18 2007 Subject: Acid Phenol/chloroform References: <1194459169.660890.101570@v3g2000hsg.googlegroups.com> Message-ID: <5pg1v6Fr22vnU1@mid.individual.net> Nick Theodorakis wrote: >Start with solid phenol crystals, and saturate it with good water (you >might need to do it a couple of times). Standard safety disclaimers >apply (wear gloves and eye and body protection, etc.). Since phenol is one of the really nasty chemicals in the molecular biology lab, I would rather buy the ready-to-use substance. Christian -- X-no-Sig: yes From ben.long from yourfinger.anu.edu.au Wed Nov 7 22:31:27 2007 From: ben.long from yourfinger.anu.edu.au (Bean Long) Date: Thu Nov 8 12:29:29 2007 Subject: Incomplete translation of N-term His-tagged protein in E.coli In-Reply-To: <47327ceb$1@clarion.carno.net.au> References: <47327ceb$1@clarion.carno.net.au> Message-ID: <47328310@clarion.carno.net.au> BTW, expression is in BL21(DE3) -- Bean Remove "yourfinger" before replying From stxsz1 from nottingham.ac.uk Thu Nov 8 12:51:18 2007 From: stxsz1 from nottingham.ac.uk (Zhong Silin) Date: Thu Nov 8 17:57:56 2007 Subject: 2 questions about kinase and phosphotase Message-ID: Hi I am going to label the 5' end of a synthetic RNA (from T7 in vitro trans). The first nucleoside (normally a G?) should has 5'-triphosphate, although I remeber some ppl told me a lot of the 5'-ppp were truncated during the in vitro trascription. question 1 Since the polynucleotide kinase PNK shouldn't be able to phosphorylate the intact RNA with 5'-triphosphate in a forward reaction, can the exchange reaction work? I know the exchange reaction can transfer a 5'-phosphate group to ADP. It should not work on the 5'-triphosphate. question 2 If neither forward nor exchange reaction can label the RNA, can I dephosphorylate it using a phosphotase (CIAP) and then label it with PNK? The same question is: can CIAP remove the triphosphate. I know it is silly to label a synthetic RNA. If I use hot NTP in the in vitro transcription, it won't need to be labeled. But it is a long story. Thanks in advance This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation. From josenet from tiscali.co.uk Thu Nov 8 20:26:23 2007 From: josenet from tiscali.co.uk (Jose de las Heras) Date: Fri Nov 9 08:11:38 2007 Subject: Acid Phenol/chloroform References: <4730CA43.90307@creighton.edu> Message-ID: <5phrp2FrbitoU1@mid.individual.net> "Dr. Hiranya S. Roychowdhury" wrote in message news:mailman.535.1194542935.23109.methods@net.bio.net... > The pH of the acid phenol is usually kept around 4.0. > We used to make it in lab with Tri.HCl, but Sigma sells the stuff for so > cheap that it doesn't pay to even equilibrate your own. Sigma sells both > Phenol and Phenol Chloroform with pH8.0 or pH4 buffers. same here, unless you need industrial amounts, the (little) extra expense is worth it to avoid hassle/handling. Jose From C.Thompson from MARLAB.AC.UK Sun Nov 11 08:48:20 2007 From: C.Thompson from MARLAB.AC.UK (Caroline Thompson) Date: Sun Nov 11 12:56:11 2007 Subject: 96-well plate DNA extraction kits Message-ID: <4DD991388A7D144EBCF4FF71A69D9FF7270FD6@FL-MAIL.marlab.ac.uk> Hi Guys, I am usually loathe to resort to commercial kit methods for DNA extraction, but working with moulluscs and local prohibitions on phenol/chloroform, it's maybe time to start. So far I have been using Qiagen spin column kits very successfully, but I need to move to a 96-well plate format. Are there any 96-well plate extraction kits available that can be spun in Sigma 2-16K or 3K15 centrifuges with 11123 / 11223 rotors and 13223 buckets? Eg Invitrogen kits can indeed be spun at the relatively slow speeds on these centrifuges (approx 2250g) - but the brochure suggests a plate height clearance of 5.75cm, and I have no idea where that is measured from, the ends of the bucket, the sides of the bucket? Any ideas? Caroline Caroline Thompson Conservation & Restoration Group FRS Freshwater Laboratory Faskally, Pitlochry, Perthshire PH16 5LB direct 01224 294462 switch 01796 472060 From stewjw from gmail.com Tue Nov 13 09:59:08 2007 From: stewjw from gmail.com (StewJW) Date: Tue Nov 13 12:49:41 2007 Subject: 96-well plate DNA extraction kits In-Reply-To: References: Message-ID: <1194965948.742492.106760@v3g2000hsg.googlegroups.com> These plates are standard SBS format and require a bucket depth of 5.75cm, therefore measure from the base of the bucket, up the side, to the top brim of the bucket. If you need any further assistance you can email: fsuk.invitrogen@thermofisher.com Best, Stewart From stewjw from gmail.com Tue Nov 13 10:03:09 2007 From: stewjw from gmail.com (StewJW) Date: Tue Nov 13 12:49:59 2007 Subject: 96-well plate DNA extraction kits In-Reply-To: <1194965948.742492.106760@v3g2000hsg.googlegroups.com> References: <1194965948.742492.106760@v3g2000hsg.googlegroups.com> Message-ID: <1194966189.493356.308570@d55g2000hsg.googlegroups.com> I assume your refering to Invitrogen's PureLink™ 96 Genomic DNA Kit, PureLink™ 96 Genomic DNA Kit K1821-04 4 × 96 preps. These plates can also be used with a vacuum manifold. From ebs15242 from creighton.edu Tue Nov 13 12:37:39 2007 From: ebs15242 from creighton.edu (Ed Siefker) Date: Tue Nov 13 13:11:36 2007 Subject: What absorbs intensely at 260nm? Message-ID: <4739E0E3.7050108@creighton.edu> I got some really strange readings on my spec. For the inverse PCR I'm planning to do I ligated 4ug of genomic DNA in a volume of 4 ml with NEB's T4 ligase and buffer. After ligation I added 400ul of NaAc and 9ml EtOH. I precipitated it, washed with 70% EtOH, air dried, and resuspended in 10mM Tris. On checking the concentration with a 1:25 dilution the readings were off the scale. I tried again with a 1:100 dilution the A260 was still extremely high it was at least readable. The concentration it gave me was 13685ng/ul and the ratios 260/230 and 260/280 were both over 3. Adding more buffer pushed those ratios up to 6. Something is clearly very wrong here. I checked some .5ug/ul lambda hindiii ladder and it read ok. What could have possibly gotten in my DNA to do this? From ucgatan from ucl.ac.uk Tue Nov 13 13:57:53 2007 From: ucgatan from ucl.ac.uk (Tom Anderson) Date: Tue Nov 13 17:36:38 2007 Subject: What absorbs intensely at 260nm? In-Reply-To: <4739E0E3.7050108@creighton.edu> References: <4739E0E3.7050108@creighton.edu> Message-ID: On Tue, 13 Nov 2007, Ed Siefker wrote: > What could have possibly gotten in my DNA to do this? Silica absorbs very strongly down there; if you used a silica-based cleanup kit anywhere in the pipeline (which you didn't, as far as i can see), that would do it. If you have a scanning spec, try running a spectrum and having a look. Silica is a big wall at ~260 and below. If you don't manage to get any sense from the spec, i guess it's time for a quantitative gel. Pain in the caudal extremity! tom -- Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT (t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) thomas.anderson@ucl.ac.uk From pow.joshi from gmail.com Tue Nov 13 15:59:34 2007 From: pow.joshi from gmail.com (Pow Joshi) Date: Tue Nov 13 17:36:48 2007 Subject: What absorbs intensely at 260nm? In-Reply-To: <4739E0E3.7050108@creighton.edu> References: <4739E0E3.7050108@creighton.edu> Message-ID: <710764ea0711131259h18657500ta57ba3075b2f208e@mail.gmail.com> On 13/11/2007, Ed Siefker wrote: > > I got some really strange readings on my spec. For the inverse > PCR I'm planning to do I ligated 4ug of genomic DNA in a volume > of 4 ml with NEB's T4 ligase and buffer. After ligation I added > 400ul of NaAc and 9ml EtOH. I precipitated it, washed with 70% > EtOH, air dried, and resuspended in 10mM Tris. just a quickie: did you use 4microgms of DNA in 4 millilitres of buffer? .... I usually do my ligations in a total of 20 microL volume, so just wondering. On checking the concentration with a 1:25 dilution the readings > were off the scale. I tried again with a 1:100 dilution the A260 > was still extremely high it was at least readable. The concentration > it gave me was 13685ng/ul and the ratios 260/230 and 260/280 were > both over 3. Adding more buffer pushed those ratios up to 6. > Something is clearly very wrong here. I checked some .5ug/ul lambda > hindiii ladder and it read ok. What could have possibly gotten in > my DNA to do this? so the concentration is actually ~13 mg/mL which is'nt so bad, is it? od 260/280: Double stranded nucleid acid typically has a ratio of 2.... 1.8 is very good. A single stranded nucleic acid/ oligo/RNA has a 260/280 ratio value ranging from 1.0 to 2.5, depending on the base content. A higher percentage of C and T bases have a lower value and those with A and G bases have a higher value. Methinks you want to check you calculations again: Also, as a precaution, allow the DNA pellet to suspend in the buffer completely.... Allow the UV lamp of the spec to warm up for at least 20 min. Blank with the buffer you are diluting the sample with. To verify your spec's fine, check another known sample. These are a few things I can think of ..... Hope that helps, Pow _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From timegg113 from gmail.com Tue Nov 13 20:37:24 2007 From: timegg113 from gmail.com (Tim Eggleston) Date: Wed Nov 14 13:30:31 2007 Subject: Cleaning and Sterilizing Incubators Message-ID: I am looking for some new ideas with regards to cleaning cell culture incubators. I have used rocal followed by a thorough wash with 70% ethanol in the past, but the lab I am currently working in does not use rocal. Will a washing with a regular non-caustic cleaner followed by a wash with 70% ethanol be good enough to rid the incubator of fungal and bacterial contaminations? Thanks! -- Tim Eggleston Horne Lab Research Assistant 2-505 Bowen Science Building University of Iowa From ebs15242 from creighton.edu Wed Nov 14 11:17:09 2007 From: ebs15242 from creighton.edu (Ed Siefker) Date: Wed Nov 14 13:30:40 2007 Subject: What absorbs intensely at 260nm? In-Reply-To: <710764ea0711131259h18657500ta57ba3075b2f208e@mail.gmail.com> References: <4739E0E3.7050108@creighton.edu> <710764ea0711131259h18657500ta57ba3075b2f208e@mail.gmail.com> Message-ID: <473B1F85.1060004@creighton.edu> Pow Joshi wrote: > > just a quickie: did you use 4microgms of DNA in 4 millilitres of buffer? > .... I usually do my ligations in a total of 20 microL volume, so just > wondering. I'm trying to do an inverse pcr which relies on intra-molecular ligations. (see http://en.wikipedia.org/wiki/Inverse_polymerase_chain_reaction) So the concentration has to be very low in order to fa > > > so the concentration is actually ~13 mg/mL which is'nt so bad, is it? Considering there were only 4ug in my entire ligation, there's no way that's actually DNA. > > od 260/280: > > Double stranded nucleid acid typically has a ratio of 2.... 1.8 is very > good. Right, a ratio lower than that might indicate some protein contamination. A ratio much, much higher than that would indicate what? > Methinks you want to check you calculations again: > Also, as a precaution, allow the DNA pellet to suspend in the buffer > completely.... > Allow the UV lamp of the spec to warm up for at least 20 min. > Blank with the buffer you are diluting the sample with. > To verify your spec's fine, check another known sample. Yes I follow these best practices. I'm not to worried about this experiment, I have more DNA I can religate for inverse pcr. I just like to know what happens when things go wrong. I suspect it might be DTT or something in the ligase buffer. The large volume I'm ligating in would mean that anything that precipitates from the buffer would be present in large amounts. Maybe I'll put some DTT in my spec today and see. -Ed From pow.joshi from gmail.com Wed Nov 14 14:13:51 2007 From: pow.joshi from gmail.com (Pow Joshi) Date: Wed Nov 14 16:00:35 2007 Subject: What absorbs intensely at 260nm? In-Reply-To: <473B1F85.1060004@creighton.edu> References: <4739E0E3.7050108@creighton.edu> <710764ea0711131259h18657500ta57ba3075b2f208e@mail.gmail.com> <473B1F85.1060004@creighton.edu> Message-ID: <710764ea0711141113v970371ah9ebc89075a0ccf6@mail.gmail.com> On 14/11/2007, Ed Siefker wrote: > > Pow Joshi wrote: > > > > just a quickie: did you use 4microgms of DNA in 4 millilitres of buffer? > > .... I usually do my ligations in a total of 20 microL volume, so just > > wondering. > > I'm trying to do an inverse pcr which relies on intra-molecular > ligations. (see > http://en.wikipedia.org/wiki/Inverse_polymerase_chain_reaction) So the > concentration has to be very low in order to fa > > > > > > > > > so the concentration is actually ~13 mg/mL which is'nt so bad, is it? > > Considering there were only 4ug in my entire ligation, there's no way > that's actually DNA. yes, of course ..... which is why I was wondering about the concentrations itself .... it is almost as if you created some DNA :-) .... So far I know, any system compound with a delocalized electronic system, can absorb in UV range 260/ 230 and 280 depending on the number of delocalised double/ triple bonds .... Organic chemists, please feel free to correct me or give additional information. Pow > > > od 260/280: > > > > Double stranded nucleid acid typically has a ratio of 2.... 1.8 is very > > good. > > Right, a ratio lower than that might indicate some protein > contamination. A ratio much, much higher than that would indicate what? > > > Methinks you want to check you calculations again: > > Also, as a precaution, allow the DNA pellet to suspend in the buffer > > completely.... > > Allow the UV lamp of the spec to warm up for at least 20 min. > > Blank with the buffer you are diluting the sample with. > > To verify your spec's fine, check another known sample. > > Yes I follow these best practices. I'm not to worried about this > experiment, I have more DNA I can religate for inverse pcr. I just like > to know what happens when things go wrong. I suspect it might be DTT or > something in the ligase buffer. The large volume I'm ligating in would > mean that anything that precipitates from the buffer would be present in > large amounts. Maybe I'll put some DTT in my spec today and see. > -Ed > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From ucgatan from ucl.ac.uk Wed Nov 14 14:23:50 2007 From: ucgatan from ucl.ac.uk (Tom Anderson) Date: Wed Nov 14 16:00:45 2007 Subject: Cleaning and Sterilizing Incubators In-Reply-To: References: Message-ID: On Tue, 13 Nov 2007, Tim Eggleston wrote: > I am looking for some new ideas with regards to cleaning cell culture > incubators. I have used rocal followed by a thorough wash with 70% > ethanol in the past, but the lab I am currently working in does not use > rocal. Will a washing with a regular non-caustic cleaner followed by a > wash with 70% ethanol be good enough to rid the incubator of fungal and > bacterial contaminations? We take the removable bits out and autoclave them, but the walls just get 70% ethanol. Seems to work. What's rocal? tom -- Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT (t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) thomas.anderson@ucl.ac.uk From amanosz from yahoo.com Wed Nov 14 21:12:58 2007 From: amanosz from yahoo.com (David Liu) Date: Wed Nov 14 23:49:57 2007 Subject: Very Low Counts with 3H Thymidine Assay / Millipore Multiscreen system Message-ID: <864790.56255.qm@web36506.mail.mud.yahoo.com> Hello, Does anyone have experience using the Millipore Multiscreen system for 3H-Thymidine incorporation assay? I am having trouble with getting very low cpm's for my assays. I get at most ~5000 cpm under proliferating conditions which is higher than no-growth controls (~2700), but nowhere near the published 20,000-50,000 cpm that I am expecting to see. I think I've narrowed it down to the cell harvesting, wash and precipitation steps, and could use any help, ideas, or suggestions. I'm using a T cell line, Kit225, which is IL-2 dependent for growth. I know they are proliferating and that my IL-2 batch is good, because I use the same conditions to maintain growth. Also, cell density is visibly higher (using microscope) under proliferating conditions at the end of 48 hours. I am following published protocols for growth and pulse times (48 hours growth, pulsing with 1uCi 3H for the final 16-18 hours). This is actually on the high end for growth and pulse times, compared to published protocols, so I should be capturing S phase and I should see 3H thymidine incorporation. I am following an old lab protocol that was used for primary T cells for the harvesting and wash steps. I am using the Multiscreen system from Millipore, with a vacuum manifold and a punching apparatus, and am using Millipore Durapore HV 0.45um filter 96 well plates. My protocol is as follows: 1) Prewet membrane, vacuum, load cells 2) Wash 2x with PBS 3) Add 5% TCA, and precipitate 20 min on ice, then vacuum filter out 4) Dry membranes, and punch into vials with 0.5mL bleach diluted 1/12 times. 5) Agitate on rotary shaker 30 minutes, then add scint fluid (Ultima gold XR) and count. I have also tried precipitating with 20% TCA, and 100% Ethanol. All wash and ppt solutions are ice cold. This is also very similar to the protocol that the Multiscreen literature recommends, though they use a different cell line, and they add ethanol washes after precipitation. As I mentioned, I'm quite confident the cells are proliferating, so I don't know where I could be going wrong. The only possibility is in step 5, which according to Millipore literature, bleach is required to release incorporated 3H from the Durapore membranes. However, the rotary shaker is not vigorously agitating (not like a tabletop vortex), but rather the speed of a shaker you would use to wash protein gels or western blots. However, I don't see how this can be causing my counts to be 10% of expected values. Does anyone have any ideas or suggestions on what I should try next or how to troubleshoot this? Thank you very much in advance! David ____________________________________________________________________________________ Get easy, one-click access to your favorites. Make Yahoo! your homepage. http://www.yahoo.com/r/hs From nobody from nospam.not Wed Nov 14 20:14:04 2007 From: nobody from nospam.not (Han) Date: Wed Nov 14 23:50:02 2007 Subject: Cleaning and Sterilizing Incubators References: Message-ID: "Tim Eggleston" wrote in news:mailman.606.1195065029.23109.methods@net.bio.net: > I am looking for some new ideas with regards to cleaning cell culture > incubators. I have used rocal followed by a thorough wash with 70% > ethanol in the past, but the lab I am currently working in does not > use rocal. Will a washing with a regular non-caustic cleaner followed > by a wash with 70% ethanol be good enough to rid the incubator of > fungal and bacterial contaminations? Thanks! > Of course nothing should grow in an incubator other then what you want to grow in dishes, flasks, or whatever. But I never thought that the insides of an incubator would be sterile (apart from what's inside the dishes, flasks, etc). The room in which the incubator stands isn't sterile, and the air in it probably isn't either. So why would you have to sterilize the walls and shelves of an incubator? -- Best regards Han email address is invalid From engelbert_buxbaum from hotmail.com Thu Nov 15 08:40:48 2007 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Thu Nov 15 13:03:52 2007 Subject: Antibody binding to nonpermeabilized cells References: Message-ID: Am 06.11.2007, 15:06 Uhr, schrieb Beug, Shawn : > I am interested in calculating the ratio of intracellular and > extracellular GFP-tagged protein in primary neurons. I am trying to > demonstrate that perturbation of posttranslational modification affects > extracellular/intracellular sublocalization. I was wondering if > immunnostaining for transfected neurons with an anti-GFP antibody Could be done. The easier way is to work with a non-membranepermeable fluorescence quencher (iodide, acrylamide or the like). The fluorescence of extracellular GFP would be quenched, that of intracellular GFP not, the ratio of fluorescence with/without quencher is, under appropriate conditions, equal to the ratio of intracellular / total GFP. See: @book{Lak-99, AUTHOR= {J.R. Lakowicz}, TITLE= {Principles of fluorescence spectroscopy}, YEAR= {1999}, PUBLISHER= {Kluwer}, ADDRESS= {New York}, EDITION= {Second}, LANGUAGE= {engl} } p.s.: It makes little sense to add a several language confidentiality statement to a message published on usenet. From engelbert_buxbaum from hotmail.com Thu Nov 15 09:15:01 2007 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Thu Nov 15 13:03:56 2007 Subject: Cleaning and Sterilizing Incubators References: Message-ID: Am 13.11.2007, 21:37 Uhr, schrieb Tim Eggleston : > I am looking for some new ideas with regards to cleaning cell culture > incubators. I have used rocal followed by a thorough wash with 70% > ethanol in the past, but the lab I am currently working in does not > use rocal. Will a washing with a regular non-caustic cleaner followed > by a wash with 70% ethanol be good enough to rid the incubator of > fungal and bacterial contaminations? Thanks! For regular maintenance 70% EtOH should do just fine. If you get frequent infections, or if the equipment was not in use for a long time I'd give it a thorough wash with Virkon-S (www.antecint.co.uk/main/virkons.htm). That stuff kills bugs without killing equipment or humans, and the smell isn't too bad either. From ucgatan from ucl.ac.uk Thu Nov 15 09:10:21 2007 From: ucgatan from ucl.ac.uk (Tom Anderson) Date: Thu Nov 15 13:04:05 2007 Subject: Cleaning and Sterilizing Incubators In-Reply-To: References: Message-ID: On Thu, 15 Nov 2007, Han wrote: > "Tim Eggleston" wrote in > news:mailman.606.1195065029.23109.methods@net.bio.net: > > > I am looking for some new ideas with regards to cleaning cell culture > > incubators. I have used rocal followed by a thorough wash with 70% > > ethanol in the past, but the lab I am currently working in does not > > use rocal. Will a washing with a regular non-caustic cleaner followed > > by a wash with 70% ethanol be good enough to rid the incubator of > > fungal and bacterial contaminations? Thanks! > > Of course nothing should grow in an incubator other then what you want > to grow in dishes, flasks, or whatever. But I never thought that the > insides of an incubator would be sterile (apart from what's inside the > dishes, flasks, etc). The room in which the incubator stands isn't > sterile, and the air in it probably isn't either. So why would you have > to sterilize the walls and shelves of an incubator? I think the goal isn't to keep the inside of the incubator sterile, it's to stop it being colonised by fungi or bacteria. There's a difference between a box full of nonsterile air, and a box full of spores - particularly if it's a warm, humid box! tom -- Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT (t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) thomas.anderson@ucl.ac.uk From hsieh_c from ccnt.norccc.usc.edu Fri Nov 16 13:45:40 2007 From: hsieh_c from ccnt.norccc.usc.edu (Chih-Lin Hsieh) Date: Fri Nov 16 15:28:40 2007 Subject: Stability Bead-Avidin-Biotin-DNA? Message-ID: Hi Jim, We are working on a project that may have a similar step as what you were doing, and I come across your posting. Have you gotten any information on how stable the streptavidin is in formamide? Did your DNA (I assume it is biotinylated) bind to the streptavidin in 64-84% formamide? Do you know whether strpetavidin becomes denatured in formamide? I am a bit puzzled by your description of white ethanol precipitate is washed off the beads when incubated with formamide. Was there a EtOH ppt step after formamide incubation or this white ppt occurs in the formamide incubation? Thanks for any information that you can share. Chih-Lin From dws from email.unc.edu Thu Nov 15 16:35:12 2007 From: dws from email.unc.edu (Darrel Stafford) Date: Fri Nov 16 15:28:51 2007 Subject: Nonfat Milk Powder & Western Blot Message-ID: <000301c827cf$6c5ce540$c30f0298@bio.unc.edu> It was from Bert o'malleys lab but I don't remember the exact reference. D Stafford From zebedeeboy from hotmail.com Mon Nov 19 05:38:09 2007 From: zebedeeboy from hotmail.com (IanMc) Date: Mon Nov 19 14:23:06 2007 Subject: Cleaning and Sterilizing Incubators References: Message-ID: "Tom Anderson" wrote in message news:mailman.614.1195074070.23109.methods@net.bio.net... > On Tue, 13 Nov 2007, Tim Eggleston wrote: > >> I am looking for some new ideas with regards to cleaning cell culture >> incubators. I have used rocal followed by a thorough wash with 70% >> ethanol in the past, but the lab I am currently working in does not use >> rocal. Will a washing with a regular non-caustic cleaner followed by a >> wash with 70% ethanol be good enough to rid the incubator of fungal and >> bacterial contaminations? > > We take the removable bits out and autoclave them, but the walls just get > 70% ethanol. Seems to work. > > What's rocal? > Benzalkonium chloride. IIRC Ian Mc > tom > > -- > Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E > 6BT > (t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) > thomas.anderson@ucl.ac.uk > From jmandresen from gmail.com Tue Nov 20 16:40:35 2007 From: jmandresen from gmail.com (Michael Andresen) Date: Tue Nov 20 17:14:38 2007 Subject: Luciferase question Message-ID: I am trying to characterize a Purkinje cell-specific promoter using Promega's dual luciferase system. Transfecting pRL-CMV into CHO cells (chosen to utilize some key stably transfected lines in the lab) gives me 10x to 20x stimulation of transcription over pRL-null. The issue is that pRL-null gives 10x stimulation over my promoter of interest. When I transfect with transcription factors known to be active in the promoter, transcription rises nicely, but all below the transcription level of the "negative control" pRL-null. I am guessing this is due to strong inhibition of the promoter by cellular factors that help restrict promoter activity to Purkinje cells. I would like to know if anyone knows of similar stories in the literature. I don't know if I should abandon the assay because of its idiosyncrasy or if the results will stand up to peer review because the luciferase assay is that sensitive. Does anyone know any references dissecting a promoter despite overall transcription levels below the negative control? Thanks, Michael Andresen From anjalirthomas from gmail.com Wed Nov 21 00:21:04 2007 From: anjalirthomas from gmail.com (Anajli Thomas) Date: Wed Nov 21 13:19:59 2007 Subject: troubleshooting + ligation Message-ID: I have been trying to clone a 1.5 kb insert to pEGFP c1 vector. The restriction enzymes used were EcoR1 and Kpn1. I have tried ligation thrice and still no colonies. JM109 was used for transformation. The restriction enzymes were added together for cutting the insert from T vector (primary clone). For ligation I added 5 ul of insert and 2ul of backbone. Could you help me out with this problem Sincerely Anjali From peterh from my.molbio.ku.dk Wed Nov 21 10:40:31 2007 From: peterh from my.molbio.ku.dk (Peter Henriksen) Date: Wed Nov 21 13:20:14 2007 Subject: Protein/protein-crosslinkers Message-ID: I am trying to design an experiment in which I will need to make protein-protein crosslinks but not DNA-protein crosslinks in living cells. In principal I think this should be possible using a crosslinker that will crosslink between two carboxylic groups but I haven't been able to find such a crosslinker so far. If anyone has knowledge of a crosslinker with the above properties I would be thrilled to know about it. Thanks Peter Henriksen From chauvaux from pasteur.fr Wed Nov 21 11:08:41 2007 From: chauvaux from pasteur.fr (Sylvie Chauvaux) Date: Wed Nov 21 13:20:20 2007 Subject: bacterial viability assay Message-ID: Hello, Does somebody know an assay to follow bacterial viability of a culture ? The commercial kits implicate to take samples of cultures. Since I need to follow the viability overnight, I need an assay where the reagents can be present in the culture without affecting growth of bacteria. Many thanks for all your ideas, Sylvie From pdeitik from bcm.tmc.edu Wed Nov 21 14:16:51 2007 From: pdeitik from bcm.tmc.edu (Deitiker, Philip R) Date: Wed Nov 21 15:05:42 2007 Subject: Protein/protein-crosslinkers In-Reply-To: Message-ID: The anhydride bond is one of the most difficult to form. In peptide synthesis the anhydride that is used to react with the amino terminal of the growing peptide is formed by carbodiimide. The anhydride of carbonyl groups is unstable and will be attacked by the lysine amino groups causing the permanent formation R-CO-N-R bond. Typically dicyclohexyl carbodiimide or diisopropyl carbodiimide. There are water soluble carbodiimied such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidic hydrochloride. Pierce cat.# 22980. More crossreactivity can be achieved by making a succinic anhydride of the protein of interest which has a higher crosslinking activity to targets. The target sites are Lysines, amino termini. This is not suitable for treatment of living cells. Carbodiimides are known as sensitizing agents, they do crosslink proteins on the skin of laboratory researches which can link gliadin (such as used to be used as a donning agent) latex and self proteins. Individuals develop dermatitis to the new antigens and this then can cause allergies to gloves, donning agents, etc. = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = There is an enzyme in living cells known as transglutaminase (tTG), it can be found in the nucleus, cytosol, and extracellular matrix. http://en.wikipedia.org/wiki/Tissue_transglutaminase http://en.wikipedia.org/wiki/Keratinocyte_transglutaminase tTG crosslinks 4 amino acid motives (for a discussion of targets review literature on gliadin as these have been the most characterized) It crosslinks the lysine amino group to glutamines and to a lessor degree apsartamine. Guinea pig transglutaminase is available, to be used the target needs to have a 4 amino acid motif that is identified by the enzyme and the enzyme will cross link other proteins to it. This will likely cause ubiquitization and targeting for degradation. -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Peter Henriksen Sent: Wednesday, November 21, 2007 9:41 AM To: methods@magpie.bio.indiana.edu Subject: Protein/protein-crosslinkers I am trying to design an experiment in which I will need to make protein-protein crosslinks but not DNA-protein crosslinks in living cells. In principal I think this should be possible using a crosslinker that will crosslink between two carboxylic groups but I haven't been able to find such a crosslinker so far. If anyone has knowledge of a crosslinker with the above properties I would be thrilled to know about it. From sudhee26 from gmail.com Wed Nov 21 14:08:14 2007 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Wed Nov 21 15:05:52 2007 Subject: troubleshooting + ligation In-Reply-To: References: Message-ID: hi, are u using any ligation kit? whats the temperature of ligation and whats the time? first thing to see is whether its ligated or not only then you should proceed towards transformation. try overnight ligation at 14 degrees and then..run an agarose gel to see your product. if its ligated it will become circular and its migration will be faster (u can see supercoiled and stuff like that) subject that again to single digestion..it will become linear and will match with new mol weight (vector+1.5kb) then try cloning it. Sudheendra. On Nov 21, 2007 10:51 AM, Anajli Thomas wrote: > I have been trying to clone a 1.5 kb insert to pEGFP c1 vector. The > restriction enzymes used were EcoR1 and Kpn1. I have tried ligation thrice > and still no colonies. JM109 was used for transformation. > > The restriction enzymes were added together for cutting the insert from T > vector (primary clone). For ligation I added 5 ul of insert and 2ul of > backbone. > > Could you help me out with this problem > > Sincerely > Anjali > _______________________________________________ > 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 sudhee26 from gmail.com Wed Nov 21 14:15:38 2007 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Wed Nov 21 15:05:59 2007 Subject: Protein/protein-crosslinkers In-Reply-To: References: Message-ID: hi, try these link http://www.protocol-online.org/biology-forums/posts/9764.html http://www.piercenet.com/Products/Browse.cfm?fldID=CE4D6C5C-5946-4814-9904-C46E01232683 https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&sku=&productDescription=8259&ref=http%3A%2F%2Fwww%2Egoogle%2Ecom%2Fsearch%3Fq%3Dprotein%2Bprotein%2Bcrosslinker%26rls%3Dcom%2Emicrosoft%3A%2A%26ie%3DUTF%2D8%26oe%3DUTF%2D8%26startIndex%3D%26startPage%3D1 I hope its of help to you Sudheendra. On Nov 21, 2007 9:10 PM, Peter Henriksen wrote: > I am trying to design an experiment in which I will need to make > protein-protein crosslinks but not DNA-protein crosslinks in living cells. > > In principal I think this should be possible using a crosslinker that will > crosslink between two carboxylic groups but I haven't been able to find such > a crosslinker so far. > > If anyone has knowledge of a crosslinker with the above properties I would > be thrilled to know about it. > > Thanks > > Peter Henriksen > > > > > _______________________________________________ > 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 josenet from tiscali.co.uk Wed Nov 21 16:56:37 2007 From: josenet from tiscali.co.uk (Jose de las Heras) Date: Wed Nov 21 17:51:23 2007 Subject: troubleshooting + ligation References: Message-ID: <5qjo6vFvo65iU1@mid.individual.net> "Anajli Thomas" wrote in message news:mailman.696.1195669227.23109.methods@net.bio.net... > I have been trying to clone a 1.5 kb insert to pEGFP c1 vector. The > restriction enzymes used were EcoR1 and Kpn1. I have tried ligation thrice > and still no colonies. JM109 was used for transformation. > > The restriction enzymes were added together for cutting the insert from T > vector (primary clone). For ligation I added 5 ul of insert and 2ul of > backbone. > > Could you help me out with this problem > > Sincerely > Anjali If somebody asked you this, do you think you have all the necessary information to be able to suggest anything helpful? You say nothing of how you purified the fragments, how teh digests looked... but what really gets to me is the "For ligation I added 5 ul of insert and 2ul of backbone". Even if I know the size of pEGFP, talking microlitres is pointless as I don't know teh concentration. It's one pet hate of mine, when people write protocols etc talking of "microlitres" as if we all knew magically what concentration of reagent they're using. It makes me think that they don't know. It makes me think that they don't really know what is important. Sorry to sound a bit harsh... but I feel it's got to be said. Just think about the problem a little bit before you post, so that you're able to give the relevant information, and I am sure you will receive more helpful answers. Regards, Jose de las Heras From wxfbmn from 163.com Thu Nov 22 01:18:06 2007 From: wxfbmn from 163.com (WANG XF) Date: Thu Nov 22 10:57:09 2007 Subject: cccDNA preparation from transfected HepG2 cells Message-ID: <404daa2f0711212218h4af18d7ew3593be5e8a73c148@mail.gmail.com> Hi, every one: Does anyone have a detailed protocol for preparing cccDNA from HepG2 cells transfected with HBV supergenome? I have tried the method of Summers, and did not get a good result. Summers method: http://jvi.asm.org/cgi/content/abstract/64/6/2819 Thank you all! LAO Wang From peterh from my.molbio.ku.dk Thu Nov 22 12:17:44 2007 From: peterh from my.molbio.ku.dk (Peter Henriksen) Date: Thu Nov 22 19:32:46 2007 Subject: Protein/protein-crosslinkers Message-ID: Thank you for your great answers. I realize that achieving the actual chemistry of crosslinking two carboxyl groups is a lot tougher than I anticipated. If I got it right, using a carbodiimide (like EDC) will be a problem for me because it prepares carboxyls to react with amines and thereby Proteins with DNA. Adding high concentrations of polyamines might out-compete DNA-amines but might also cause very little crosslinking since carboxyl groups have to bind the same polyamine-molecule to be crosslinked. The use of more target-specifik crosslinking methods (like transglutaminase under a controlled promoter) seems less attractive to me because I want the crosslinking to be fairly unspecifik and capable of crossbinding lots of different proteins. However, knowing that I have to rethink my strategy is also of great value. :o) Cheers Peter Henriksen From metugenetics from yahoo.com Thu Nov 22 15:00:07 2007 From: metugenetics from yahoo.com (Ozan Aygun) Date: Thu Nov 22 19:32:52 2007 Subject: Protein/protein-crosslinkers Message-ID: <608483.91501.qm@web90410.mail.mud.yahoo.com> Hi Peter, Some years ago I have used a crosslinking reagent called as DSP. This should be available from Pierce. It is usually quite good when used properly, and thiol cleavable. I am not quite sure whether it fits your requirements but worth to try. There are even more crosslinkers offered by Pierce, perhaps you should look into their web catalog for properties. Best, Ozan ____________________________________________________________________________________ Get easy, one-click access to your favorites. Make Yahoo! your homepage. http://www.yahoo.com/r/hs From pow.joshi from gmail.com Thu Nov 22 16:11:32 2007 From: pow.joshi from gmail.com (Pow Joshi) Date: Thu Nov 22 19:32:58 2007 Subject: bacterial viability assay In-Reply-To: References: Message-ID: <710764ea0711221311o16b42312ocb7aa0e0952fa982@mail.gmail.com> On 21/11/2007, Sylvie Chauvaux wrote: > > Hello, > > Does somebody know an assay to follow bacterial viability of a > culture ? The commercial kits implicate to take samples of cultures. > Since I need to follow the viability overnight, I need an assay where > the reagents can be present in the culture without affecting growth > of bacteria. > > Many thanks for all your ideas, I don't claim to know too much about microbes, however would'nt plating the bugs either on regular LB or mininal medum work? Ofcourse LB would need to have an antibiotic marker, I guess. Pow Sylvie > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From virashkgupta from gmail.com Thu Nov 22 23:21:32 2007 From: virashkgupta from gmail.com (Virash Gupta) Date: Fri Nov 23 13:28:59 2007 Subject: Methods Digest, Vol 30, Issue 14-Bacterial viability assay Message-ID: Bacterial viability assay: You need to write specific experiment-name of bacteria, cultural conditions and what you want to test, for correct suggestions. V K Gupta Sr Microbiologist Insect Mol Biology Lab PAU On 11/22/07, 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. troubleshooting + ligation (Anajli Thomas) > 2. bacterial viability assay (Sylvie Chauvaux) > 3. Protein/protein-crosslinkers (Peter Henriksen) > 4. RE: Protein/protein-crosslinkers (Deitiker, Philip R) > 5. Re: troubleshooting + ligation (Sudheendra Rao N R) > 6. Re: Protein/protein-crosslinkers (Sudheendra Rao N R) > 7. Re: troubleshooting + ligation (Jose de las Heras) > 8. cccDNA preparation from transfected HepG2 cells (WANG XF) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 21 Nov 2007 10:51:04 +0530 > From: "Anajli Thomas" > Subject: troubleshooting + ligation > To: methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > I have been trying to clone a 1.5 kb insert to pEGFP c1 vector. The > restriction enzymes used were EcoR1 and Kpn1. I have tried ligation thrice > and still no colonies. JM109 was used for transformation. > > The restriction enzymes were added together for cutting the insert from T > vector (primary clone). For ligation I added 5 ul of insert and 2ul of > backbone. > > Could you help me out with this problem > > Sincerely > Anjali > > > ------------------------------ > > Message: 2 > Date: Wed, 21 Nov 2007 17:08:41 +0100 > From: Sylvie Chauvaux > Subject: bacterial viability assay > To: methods@magpie.bio.indiana.edu > Message-ID: > Content-Type: text/plain; charset="us-ascii" ; format="flowed" > > Hello, > > Does somebody know an assay to follow bacterial viability of a > culture ? The commercial kits implicate to take samples of cultures. > Since I need to follow the viability overnight, I need an assay where > the reagents can be present in the culture without affecting growth > of bacteria. > > Many thanks for all your ideas, > > Sylvie > > > > ------------------------------ > > Message: 3 > Date: Wed, 21 Nov 2007 16:40:31 +0100 > From: "Peter Henriksen" > Subject: Protein/protein-crosslinkers > To: > Message-ID: > > Content-Type: text/plain; charset="iso-8859-1" > > I am trying to design an experiment in which I will need to make protein-protein crosslinks but not DNA-protein crosslinks in living cells. > > In principal I think this should be possible using a crosslinker that will crosslink between two carboxylic groups but I haven't been able to find such a crosslinker so far. > > If anyone has knowledge of a crosslinker with the above properties I would be thrilled to know about it. > > Thanks > > Peter Henriksen > > > > > > > ------------------------------ > > Message: 4 > Date: Wed, 21 Nov 2007 13:16:51 -0600 > From: "Deitiker, Philip R" > Subject: RE: Protein/protein-crosslinkers > To: > Message-ID: > > Content-Type: text/plain; charset="us-ascii" > > > > The anhydride bond is one of the most difficult to form. > > In peptide synthesis the anhydride that is used to react with the amino > terminal of the growing peptide is formed by carbodiimide. The anhydride of > carbonyl groups is unstable and will be attacked by the lysine amino groups causing the permanent formation R-CO-N-R bond. > > Typically dicyclohexyl carbodiimide or diisopropyl carbodiimide. > There are water soluble carbodiimied such as > 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidic hydrochloride. > Pierce cat.# 22980. More crossreactivity can be achieved by making a succinic > anhydride of the protein of interest which has a higher crosslinking activity > to targets. The target sites are Lysines, amino termini. > > This is not suitable for treatment of living cells. Carbodiimides are known as sensitizing agents, they do crosslink proteins on the skin of laboratory researches which can link gliadin (such as used to be used as a donning agent) latex and self proteins. Individuals develop dermatitis to the new > antigens and this then can cause allergies to gloves, donning agents, etc. > > = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = > > There is an enzyme in living cells known as transglutaminase (tTG), > it can be found in the nucleus, cytosol, and extracellular matrix. > > http://en.wikipedia.org/wiki/Tissue_transglutaminase > http://en.wikipedia.org/wiki/Keratinocyte_transglutaminase > > tTG crosslinks 4 amino acid motives (for a discussion of targets > review literature on gliadin as these have been the most characterized) > It crosslinks the lysine amino group to glutamines and to a lessor degree apsartamine. > Guinea pig transglutaminase is available, to be used the target needs to have a 4 amino acid motif that is identified by the enzyme and the enzyme will cross link other proteins to it. This will likely cause ubiquitization and targeting for degradation. > > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Peter Henriksen > Sent: Wednesday, November 21, 2007 9:41 AM > To: methods@magpie.bio.indiana.edu > Subject: Protein/protein-crosslinkers > > I am trying to design an experiment in which I will need to make protein-protein crosslinks but not DNA-protein crosslinks in living cells. > > In principal I think this should be possible using a crosslinker that will crosslink between two carboxylic groups but I haven't been able to find such a crosslinker so far. > > > If anyone has knowledge of a crosslinker with the above properties I would be thrilled to know about it. > > > > > > ------------------------------ > > Message: 5 > Date: Thu, 22 Nov 2007 00:38:14 +0530 > From: "Sudheendra Rao N R" > Subject: Re: troubleshooting + ligation > To: "Anajli Thomas" , > Methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > hi, > are u using any ligation kit? > whats the temperature of ligation and whats the time? > first thing to see is whether its ligated or not > only then you should proceed towards transformation. > try overnight ligation at 14 degrees and then..run an agarose gel to see > your product. > if its ligated it will become circular and its migration will be faster (u > can see supercoiled and stuff like that) > subject that again to single digestion..it will become linear and will match > with new mol weight (vector+1.5kb) > then try cloning it. > > Sudheendra. > > On Nov 21, 2007 10:51 AM, Anajli Thomas wrote: > > > I have been trying to clone a 1.5 kb insert to pEGFP c1 vector. The > > restriction enzymes used were EcoR1 and Kpn1. I have tried ligation thrice > > and still no colonies. JM109 was used for transformation. > > > > The restriction enzymes were added together for cutting the insert from T > > vector (primary clone). For ligation I added 5 ul of insert and 2ul of > > backbone. > > > > Could you help me out with this problem > > > > Sincerely > > Anjali > > _______________________________________________ > > 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 > > > ------------------------------ > > Message: 6 > Date: Thu, 22 Nov 2007 00:45:38 +0530 > From: "Sudheendra Rao N R" > Subject: Re: Protein/protein-crosslinkers > To: "Peter Henriksen" , > methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > hi, > try these link > http://www.protocol-online.org/biology-forums/posts/9764.html > > http://www.piercenet.com/Products/Browse.cfm?fldID=CE4D6C5C-5946-4814-9904-C46E01232683 > > https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&sku=&productDescription=8259&ref=http%3A%2F%2Fwww%2Egoogle%2Ecom%2Fsearch%3Fq%3Dprotein%2Bprotein%2Bcrosslinker%26rls%3Dcom%2Emicrosoft%3A%2A%26ie%3DUTF%2D8%26oe%3DUTF%2D8%26startIndex%3D%26startPage%3D1 > > > I hope its of help to you > > Sudheendra. > On Nov 21, 2007 9:10 PM, Peter Henriksen wrote: > > > I am trying to design an experiment in which I will need to make > > protein-protein crosslinks but not DNA-protein crosslinks in living cells. > > > > In principal I think this should be possible using a crosslinker that will > > crosslink between two carboxylic groups but I haven't been able to find such > > a crosslinker so far. > > > > If anyone has knowledge of a crosslinker with the above properties I would > > be thrilled to know about it. > > > > Thanks > > > > Peter Henriksen > > > > > > > > > > _______________________________________________ > > 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 > > > ------------------------------ > > Message: 7 > Date: Wed, 21 Nov 2007 21:56:37 -0000 > From: "Jose de las Heras" > Subject: Re: troubleshooting + ligation > To: methods@net.bio.net > Message-ID: <5qjo6vFvo65iU1@mid.individual.net> > > > "Anajli Thomas" wrote in message > news:mailman.696.1195669227.23109.methods@net.bio.net... > > I have been trying to clone a 1.5 kb insert to pEGFP c1 vector. The > > restriction enzymes used were EcoR1 and Kpn1. I have tried ligation thrice > > and still no colonies. JM109 was used for transformation. > > > > The restriction enzymes were added together for cutting the insert from T > > vector (primary clone). For ligation I added 5 ul of insert and 2ul of > > backbone. > > > > Could you help me out with this problem > > > > Sincerely > > Anjali > > If somebody asked you this, do you think you have all the necessary > information to be able to suggest anything helpful? > > You say nothing of how you purified the fragments, how teh digests looked... > but what really gets to me is the "For ligation I added 5 ul of insert and > 2ul of backbone". Even if I know the size of pEGFP, talking microlitres is > pointless as I don't know teh concentration. > > It's one pet hate of mine, when people write protocols etc talking of > "microlitres" as if we all knew magically what concentration of reagent > they're using. It makes me think that they don't know. It makes me think > that they don't really know what is important. > > Sorry to sound a bit harsh... but I feel it's got to be said. > > Just think about the problem a little bit before you post, so that you're > able to give the relevant information, and I am sure you will receive more > helpful answers. > > Regards, > > Jose de las Heras > > > > > ------------------------------ > > Message: 8 > Date: Thu, 22 Nov 2007 14:18:06 +0800 > From: "WANG XF" > Subject: cccDNA preparation from transfected HepG2 cells > To: methods@magpie.bio.indiana.edu > Message-ID: > <404daa2f0711212218h4af18d7ew3593be5e8a73c148@mail.gmail.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Hi, every one: > > Does anyone have a detailed protocol for preparing cccDNA from HepG2 cells > transfected with HBV supergenome? I have tried the method of Summers, and > did not get a good result. > > Summers method: > http://jvi.asm.org/cgi/content/abstract/64/6/2819 > > Thank you all! > > LAO Wang > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 30, Issue 14 > *************************************** > -- 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 Fri Nov 23 13:12:18 2007 From: virashkgupta from gmail.com (Virash Gupta) Date: Fri Nov 23 13:29:06 2007 Subject: Methods Digest, Vol 30, Issue 15 In-Reply-To: <200711231704.lANH45Y12692@net.bio.net> References: <200711231704.lANH45Y12692@net.bio.net> Message-ID: For testing viability of culture, you need to explain the organism and your experimental requirements. Different persons and similarly different microbes act differently in different circumstances. V K gupta Prof Microbiology Spec: Mol Biology On Nov 23, 2007 10:34 PM, 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/protein-crosslinkers (Peter Henriksen) > 2. RE:Protein/protein-crosslinkers (Ozan Aygun) > 3. Re: bacterial viability assay (Pow Joshi) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Thu, 22 Nov 2007 18:17:44 +0100 > From: "Peter Henriksen" > Subject: RE: Protein/protein-crosslinkers > To: > Message-ID: > > Content-Type: text/plain; charset="iso-8859-1" > > Thank you for your great answers. > > I realize that achieving the actual chemistry of crosslinking two carboxyl groups is a lot tougher than I anticipated. > > If I got it right, using a carbodiimide (like EDC) will be a problem for me because it prepares carboxyls to react with amines and thereby Proteins with DNA. Adding high concentrations of polyamines might out-compete DNA-amines but might also cause very little crosslinking since carboxyl groups have to bind the same polyamine-molecule to be crosslinked. > > The use of more target-specifik crosslinking methods (like transglutaminase under a controlled promoter) seems less attractive to me because I want the crosslinking to be fairly unspecifik and capable of crossbinding lots of different proteins. > > However, knowing that I have to rethink my strategy is also of great value. :o) > > Cheers > > Peter Henriksen > > > > > ------------------------------ > > Message: 2 > Date: Thu, 22 Nov 2007 12:00:07 -0800 (PST) > From: Ozan Aygun > Subject: RE:Protein/protein-crosslinkers > To: methods@magpie.bio.indiana.edu > Message-ID: <608483.91501.qm@web90410.mail.mud.yahoo.com> > Content-Type: text/plain; charset=iso-8859-1 > > Hi Peter, > > Some years ago I have used a crosslinking reagent > called as DSP. This should be available from Pierce. > It is usually quite good when used properly, and thiol > cleavable. > > I am not quite sure whether it fits your requirements > but worth to try. There are even more crosslinkers > offered by Pierce, perhaps you should look into their > web catalog for properties. > > Best, > > Ozan > > > ____________________________________________________________________________________ > Get easy, one-click access to your favorites. > Make Yahoo! your homepage. > http://www.yahoo.com/r/hs > > > > ------------------------------ > > Message: 3 > Date: Thu, 22 Nov 2007 16:11:32 -0500 > From: "Pow Joshi" > Subject: Re: bacterial viability assay > To: "Sylvie Chauvaux" > Cc: methods@magpie.bio.indiana.edu > Message-ID: > <710764ea0711221311o16b42312ocb7aa0e0952fa982@mail.gmail.com> > Content-Type: text/plain; charset=ISO-8859-1 > > On 21/11/2007, Sylvie Chauvaux wrote: > > > > Hello, > > > > Does somebody know an assay to follow bacterial viability of a > > culture ? The commercial kits implicate to take samples of cultures. > > Since I need to follow the viability overnight, I need an assay where > > the reagents can be present in the culture without affecting growth > > of bacteria. > > > > Many thanks for all your ideas, > > > > > > I don't claim to know too much about microbes, however would'nt plating the > bugs either on regular LB or mininal medum work? Ofcourse LB would need to > have an antibiotic marker, I guess. > > Pow > > Sylvie > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 30, Issue 15 > *************************************** > -- 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 engelbert_buxbaum from hotmail.com Mon Nov 26 07:03:09 2007 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Nov 26 10:07:17 2007 Subject: Protein/protein-crosslinkers References: Message-ID: Am 21.11.2007, 11:40 Uhr, schrieb Peter Henriksen : > I am trying to design an experiment in which I will need to make > protein-protein crosslinks but not DNA-protein crosslinks in living > cells. The most simple chemistry to use would be those reagents that cross-link SH groups. The best source for info is probably the Molecular Probes catalogue www.probes.com. If that is insufficient, try @book{Lun-05, AUTHOR= {R.L. Lundblad}, TITLE= {Chemical reagents for protein modification}, YEAR= {2005}, PUBLISHER= {CRC Press}, ADDRESS= {Boca Raton}, EDITION= {3}, ISBN= {0-8493-1983-8}, PRICE= {129.95 US\$}, LANGUAGE= {engl} } From grtnp from yahoo.com Mon Nov 26 21:02:34 2007 From: grtnp from yahoo.com (Dhruba Tripathi) Date: Mon Nov 26 22:24:19 2007 Subject: Arabidopsis plasmid rescue In-Reply-To: Message-ID: <200313.21548.qm@web60411.mail.yahoo.com> Hello, I activation tagged the arabidopsis plant (Col-0) by vector pSKI015. First I used TAIL-PCR with all possible primers AD and 3 nested TDNA left border primers as described by Liu et at. 1995. But I failed many times in TAIL PCR with some interesting mutants . After then I tried for plasmid rescue with EcoRI for RB and Bam HI, SpeI for LB. In plasmid rescue, I got some (10-20) colonies per plate. This is more less numbers as than described by other authors. However, further I tested for reference by striking on LB plates containing 100mg/L amp plates, colonies were grew well and then subcultured it. The cell growth in amp containing media is excellent like pSKI015 vector. Then, I isolated Plasmid DNA by using alkaline lysis method and QIAGEN plasmid kit as well. But the Plasmid DNA precipitation is very low (it is impossible for digestion and sequencing). I also did midi prep the result is same. Do you have any ideas, How to isolate enough pDNA from the plasmid rescued Ecoli cells. Thank you in advance for your kind suggestion Sincerely yours Giri Tripathi Korea --------------------------------- Get easy, one-click access to your favorites. Make Yahoo! your homepage. From sadresm2002 from yahoo.com Wed Nov 28 06:54:45 2007 From: sadresm2002 from yahoo.com (Esmaeil Sadroddiny) Date: Wed Nov 28 14:33:49 2007 Subject: sequence of polymeric proteins Message-ID: <62247.35542.qm@web32607.mail.mud.yahoo.com> Hi I am working on polymeric proteins and I need to find sequence data about all chains of protein in data basis. Dose anybody knows which data basis is the best. Regards Sadroddiny.E --------------------------------- Get easy, one-click access to your favorites. Make Yahoo! your homepage. From R.Jayakumar from roswellpark.org Thu Nov 29 09:21:28 2007 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Thu Nov 29 13:10:03 2007 Subject: sequence of polymeric proteins In-Reply-To: <62247.35542.qm@web32607.mail.mud.yahoo.com> Message-ID: <97101976F8A044468CA74FE11883B90E173E7932@VISTA.roswellpark.org> You mean database. There are several protein databases on the web. You could start with www.ncbi.nlm.nih.gov. Also check out http://au.expasy.org for more protein links. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Esmaeil Sadroddiny Sent: Wednesday, November 28, 2007 6:55 AM To: methods@magpie.bio.indiana.edu Subject: sequence of polymeric proteins Hi I am working on polymeric proteins and I need to find sequence data about all chains of protein in data basis. Dose anybody knows which data basis is the best. Regards Sadroddiny.E --------------------------------- Get easy, one-click access to your favorites. Make Yahoo! your homepage. _______________________________________________ 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 vadivelarunachalam from yahoo.com Thu Nov 29 04:06:58 2007 From: vadivelarunachalam from yahoo.com (V Arunachalam) Date: Thu Nov 29 13:22:23 2007 Subject: speI xbaI sublconing Message-ID: <491113.70540.qm@web36614.mail.mud.yahoo.com> Hi, I am facing problems in cohesive subcloning a 500 bp insert from the double digest of SpeI and XbaI onto a vector of 13.5 Kb size at the same sites. Please suggest me is it correct strategy as these two enzymes generate similar ends though differ in recognition sites. ____________________________________________________________________________________ Never miss a thing. Make Yahoo your home page. http://www.yahoo.com/r/hs From zineldeen from gmail.com Fri Nov 30 20:54:26 2007 From: zineldeen from gmail.com (doaa zineldeen) Date: Fri Nov 30 21:23:13 2007 Subject: Methods Digest, Vol 30, Issue 20 In-Reply-To: <200711301703.lAUH3lY12228@net.bio.net> References: <200711301703.lAUH3lY12228@net.bio.net> Message-ID: <440923e10711301754i60d94e4bk8cf4f515bcfef537@mail.gmail.com> hello I am having problem in detection of my protein which is a 72 KD kinase by western blotting I am using a commercial one , it could detect my kinase by IF howevere on western blotting many faint bands are seen and high background I tried different blocking and different dilutions of the primary and secondary and increasedwashing time but no clear band anyone can help thank you