From engelbert_buxbaum from hotmail.com Mon Dec 1 07:34:10 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Dec 1 12:08:45 2008 Subject: protocol for liposome preparation References: Message-ID: Am 29.11.2008, 14:16 Uhr, schrieb michael nelson : > I have some difficulties preparing for clear liposomes solutions. Here > is what I did, please advise. > > ?I start off with 0.1g/ml phosphatidylcholine chloroform solution. I > then move it into a round bottomed flask and dry it in a rotary > evaporator. However, instead of getting thin films on the surface, what > I end up with is a large chunk of light yellowish solid. Does that mean > I have to use a larger volume of organic solvent? > > I then dissolve them into buffer and sonicate. Finally, I got some milky > suspension. I assume they are multimellar vesicles. My advisor told me > that I could sonicate to make them unilamellar and make the solution > goes clear. I tried, but didn't work. There is the classical method of pressing these multilamellar vesicles through a narrow-pore membrane. However, more consistent results you will obtain by dissolving the lipids with detergents as mixed micelles and then striping the detergent away with polystyrene beads (e.g. Biobeads SM2). Rigaud's group has done extensive work on this, check their papers. From biocjh from gmail.com Wed Dec 3 09:18:02 2008 From: biocjh from gmail.com (jh) Date: Wed Dec 3 12:52:15 2008 Subject: protocol for liposome preparation In-Reply-To: <0f3b6406-03cb-44c1-b7f1-81853b440f99@v4g2000yqa.googlegroups.com> References: <0f3b6406-03cb-44c1-b7f1-81853b440f99@v4g2000yqa.googlegroups.com> Message-ID: Excuse me, may you explain more detail for "PC". Whether the liposome is prepared for cell transfection experiment? Jianhao 2008/11/30 WS : > Hi Michael, > > you might try this: dissolve PC in EtOH and inject into 20 to 50 fold > volume of buffer with an insulin syringe (or the narrowest needle you > can get) while vortexing. If the EtOH might cause problems downstream, > dialyze against buffer. > > Worked well for PC/Ceramide vesicles for me. > > best regards, > > Wo > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From allison from nospam.com Wed Dec 3 12:18:47 2008 From: allison from nospam.com (Allison) Date: Wed Dec 3 18:51:18 2008 Subject: Mouse age for hybridoma fusions? Message-ID: <49368540$0$5549$9a6e19ea@news.newshosting.com> In a project to make monoclonal antibodies the standard procedure is to start with mice 4-6 weeks old, do a second boost after about 3 weeks, then, if the titer is good, go ahead with a final boost and fusion. This means that the mice are about 10-12 weeks old at the time of fusion. However, sometimes a fusion doesn't work, or the resulting monoclonal antibodies don't have quite the reactivity that one wants, and subsequent fusions are called for. Since I usually immunize 5 mice at a time and only use 1 or 2 in a first fusion I always have "spare" mice that could potentially be boosted for a second set of fusions. It would save a lot of time if I didn't have to start from scratch with new mice but would only be worthwhile if I had a reasonable fusion efficiency. So my question is: at what age are immunized mice too old to use for fusions? Does anyone have practical experience with this? TIA Allison From novalidaddress from nurfuerspam.de Wed Dec 3 16:00:58 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Dec 3 18:51:22 2008 Subject: protocol for liposome preparation References: <0f3b6406-03cb-44c1-b7f1-81853b440f99@v4g2000yqa.googlegroups.com> Message-ID: <87c6bdc4-6160-4989-bde3-01ba05af37a0@j11g2000yqg.googlegroups.com> On 3 Dez., 15:18, jh wrote: > Excuse me, may you explain more detail for "PC". > Whether the liposome is prepared for cell transfection experiment? > > Jianhao > > 2008/11/30 WS : > > > Hi Michael, Hi, PC means phosphatidylcholine. I haven't used them for transfection but to transfer a fluorescently labeled ceramid into fibroblasts. Regards, Wo > > you might try this: dissolve PC in EtOH and inject into 20 to 50 fold > > volume of buffer with an insulin syringe (or the narrowest needle you > > can get) while vortexing. If the EtOH might cause problems downstream, > > dialyze against buffer. > > > Worked well for PC/Ceramide vesicles for me. > > > best regards, > > > Wo > > _______________________________________________ > > Methods mailing list > > Meth...@net.bio.net > >http://www.bio.net/biomail/listinfo/methods From biocjh from gmail.com Thu Dec 4 21:26:11 2008 From: biocjh from gmail.com (jh) Date: Thu Dec 4 23:10:26 2008 Subject: protocol for liposome preparation In-Reply-To: <87c6bdc4-6160-4989-bde3-01ba05af37a0@j11g2000yqg.googlegroups.com> References: <0f3b6406-03cb-44c1-b7f1-81853b440f99@v4g2000yqa.googlegroups.com> <87c6bdc4-6160-4989-bde3-01ba05af37a0@j11g2000yqg.googlegroups.com> Message-ID: Thanks. I wonder whether anyone prepare liposome by self for cell transfection experiment?Cause ,I thought, this may help for someone who do not have much fund. rgds? jianhao 2008/12/4, WS : > On 3 Dez., 15:18, jh wrote: > > Excuse me, may you explain more detail for "PC". > > Whether the liposome is prepared for cell transfection experiment? > > > > Jianhao > > > > 2008/11/30 WS : > > > > > Hi Michael, > Hi, > > PC means phosphatidylcholine. I haven't used them for transfection but > to transfer a fluorescently labeled ceramid into fibroblasts. > > Regards, > > Wo > > > > > you might try this: dissolve PC in EtOH and inject into 20 to 50 fold > > > volume of buffer with an insulin syringe (or the narrowest needle you > > > can get) while vortexing. If the EtOH might cause problems downstream, > > > dialyze against buffer. > > > > > Worked well for PC/Ceramide vesicles for me. > > > > > best regards, > > > > > Wo > > > _______________________________________________ > > > Methods mailing list > > > Meth...@net.bio.net > > >http://www.bio.net/biomail/listinfo/methods > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From biocjh from gmail.com Sat Dec 6 05:10:38 2008 From: biocjh from gmail.com (jh) Date: Sat Dec 6 12:49:02 2008 Subject: protocol for liposome preparation In-Reply-To: References: <0f3b6406-03cb-44c1-b7f1-81853b440f99@v4g2000yqa.googlegroups.com> <87c6bdc4-6160-4989-bde3-01ba05af37a0@j11g2000yqg.googlegroups.com> Message-ID: thanks Do you have the protocol in detail? Or Would you like to recommendate one? rgds! Jh 2008/12/5, DK : > In article , jh wrote: > >Thanks. > >I wonder whether anyone prepare liposome by self for cell transfection > >experiment?Cause ,I thought, this may help for someone who do not have > >much fund. > > People who don't have funds to afford various lipofection reagents > on the market would probably do well by carefully optimizing > polyethyleneimine-mediated transfection. Lots of reports that suggest > that PEI gives transfection efficiencies comparable to many cationic > lipids. > > DK > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From chamosilencio from yahoo.com Sun Dec 7 21:43:58 2008 From: chamosilencio from yahoo.com (Nicosnake) Date: Sun Dec 7 22:37:00 2008 Subject: PAGE gel sticked on both plates Message-ID: <20888761.post@talk.nabble.com> HI everyone, i've been having this problem whit my acrylamide gels: after acrylamide is polymerized between the two glass plates it's really hard to separate both plates and after a long effort i can take them appart only to realize that the gel has sticked to BOTH plates, the gel is actually cut in half. I treat surfaces of both plates, one with bind silane (so the acrylamide sticks to this one) and the other with Sigmacote to prevent the gel from sticking. But still the gel sticks to both surfaces and after i separate the glases it's useless. Has anyonehad this kind of trouble?? ANy suggestion is accepted. -- View this message in context: http://www.nabble.com/PAGE-gel-sticked-on-both-plates-tp20888761p20888761.html Sent from the Bio.net - Methods mailing list archive at Nabble.com. From biocjh from gmail.com Mon Dec 8 02:50:11 2008 From: biocjh from gmail.com (jh) Date: Mon Dec 8 12:17:38 2008 Subject: PAGE gel sticked on both plates In-Reply-To: <20888761.post@talk.nabble.com> References: <20888761.post@talk.nabble.com> Message-ID: how about trying to soak the gel and plates in buffer for seperation 2008/12/8 Nicosnake : > > HI everyone, i've been having this problem whit my acrylamide gels: after > acrylamide is polymerized between the two glass plates it's really hard to > separate both plates and after a long effort i can take them appart only to > realize that the gel has sticked to BOTH plates, the gel is actually cut in > half. I treat surfaces of both plates, one with bind silane (so the > acrylamide sticks to this one) and the other with Sigmacote to prevent the > gel from sticking. But still the gel sticks to both surfaces and after i > separate the glases it's useless. Has anyonehad this kind of trouble?? ANy > suggestion is accepted. > -- > View this message in context: http://www.nabble.com/PAGE-gel-sticked-on-both-plates-tp20888761p20888761.html > Sent from the Bio.net - Methods mailing list archive at Nabble.com. > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From parbou1 from lsu.edu Mon Dec 8 10:41:16 2008 From: parbou1 from lsu.edu (Patricia C Arbour-Reily) Date: Mon Dec 8 12:17:56 2008 Subject: PAGE gel sticked on both plates In-Reply-To: <20888761.post@talk.nabble.com> References: <20888761.post@talk.nabble.com> Message-ID: <731CECC23FB8CA4E9127BD399744D1EC01406216@email001.lsu.edu> 1) I have had other brands of "Sigmacote" go bad......2)Rain-X , for your car windows , works OK but you must treat more often...its a bit more green and will do in a pinch while you replace Sigmacote , if gone bad. 3) We have had plates get gunk on them from something in the wash up...one time, a student ran all plates thru a dishwasher and used the correct soap for a dishwasher but the wrong soap for the plates , obviously...... I don't know how that was corrected Pat -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Nicosnake Sent: Sunday, December 07, 2008 8:44 PM To: methods@magpie.bio.indiana.edu Subject: PAGE gel sticked on both plates HI everyone, i've been having this problem whit my acrylamide gels: after acrylamide is polymerized between the two glass plates it's really hard to separate both plates and after a long effort i can take them appart only to realize that the gel has sticked to BOTH plates, the gel is actually cut in half. I treat surfaces of both plates, one with bind silane (so the acrylamide sticks to this one) and the other with Sigmacote to prevent the gel from sticking. But still the gel sticks to both surfaces and after i separate the glases it's useless. Has anyonehad this kind of trouble?? ANy suggestion is accepted. -- View this message in context: http://www.nabble.com/PAGE-gel-sticked-on-both-plates-tp20888761p2088876 1.html Sent from the Bio.net - Methods mailing list archive at Nabble.com. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From vasipalli from mac.com Tue Dec 9 12:17:44 2008 From: vasipalli from mac.com (Mahesh Reddy) Date: Tue Dec 9 17:26:19 2008 Subject: PAGE gel sticked on both plates In-Reply-To: <20888761.post@talk.nabble.com> References: <20888761.post@talk.nabble.com> Message-ID: <20919226.post@talk.nabble.com> Hi When u use plates first time..u need to wash the plates with NaOH.Wash the plates with 100% Etoh before making the gel.After making gel..store in Fridge until you run the gel.you can store the gel 20 days in fridge. If u still getting problem with plates..u have to check ur buffer s. I never get any problem with my plates. Best wishes Mahesh Nicosnake wrote: > > HI everyone, i've been having this problem whit my acrylamide gels: after > acrylamide is polymerized between the two glass plates it's really hard to > separate both plates and after a long effort i can take them appart only > to realize that the gel has sticked to BOTH plates, the gel is actually > cut in half. I treat surfaces of both plates, one with bind silane (so the > acrylamide sticks to this one) and the other with Sigmacote to prevent the > gel from sticking. But still the gel sticks to both surfaces and after i > separate the glases it's useless. Has anyonehad this kind of trouble?? ANy > suggestion is accepted. > -- View this message in context: http://www.nabble.com/PAGE-gel-sticked-on-both-plates-tp20888761p20919226.html Sent from the Bio.net - Methods mailing list archive at Nabble.com. From chamosilencio from yahoo.com Tue Dec 9 13:32:46 2008 From: chamosilencio from yahoo.com (Nicosnake) Date: Tue Dec 9 17:26:25 2008 Subject: PAGE gel sticked on both plates In-Reply-To: <20888761.post@talk.nabble.com> References: <20888761.post@talk.nabble.com> Message-ID: <20920660.post@talk.nabble.com> Hello everybody, thanks for all your advice and recommendations. Fortunately, the sticking problem seems to be solved. After posting the original message, i tried treating both plates with Sigmacote, to see if the bind silane was the problem. I expected the gel separate from both plates but surprisingly it stayed sticked to the one that had been previously treated with bind silane. So i guess, as one of you said it might have been too much of bind silane. I'll folow the recommendation of coating just one plate with sigmacote and leaving the other one "clean" so the gel sticks to this one. Thanks for all your help, i really appreaciate it. -- View this message in context: http://www.nabble.com/PAGE-gel-sticked-on-both-plates-tp20888761p20920660.html Sent from the Bio.net - Methods mailing list archive at Nabble.com. From rao.ih from brunet.bn Wed Dec 10 03:10:40 2008 From: rao.ih from brunet.bn (Dr Rao) Date: Wed Dec 10 11:50:44 2008 Subject: Help - diagnostic PCR lab layout Message-ID: <000001c95a9e$cb15dcb0$0801a8c0@IDEALE25114231> Dear Sir, Good wishes to you. We have a poultry farm with Broiler hatchery, Commercial broilers and layers in Brunei. We are planning to have a small diagnostic laboratory for our poultry farm to identify infectious bacteria and viruses like IBD, IB, ND, ILT, CAV. Also need to test Antibiotic sensitivity in our Poultry farm. Water testing is also required. Please advise and suggest. Thanks and regards. Dr. Rao From J-R.Kraehling from tu-braunschweig.de Wed Dec 10 05:50:31 2008 From: J-R.Kraehling from tu-braunschweig.de (=?iso-8859-1?Q?Jan_R._Kr=E4hling?=) Date: Wed Dec 10 11:50:50 2008 Subject: Size-Exclusion Message-ID: <5901645BB669488DBFF736FCC311D125@Jan> Hello! We are trying to establish the sixe-exclusion chromatography in our lab to purify the enzyme of our interest. In the purification-steps before the size exclusion we observe that a NaCl-concentration of 1M stabilize the enzyme better than lower NaCl concentrations. So we tried the size exclusion at first with a high salt concentration, but the enzyme wasn't detectable. At lower concentrations the enzyme is detectable and active. My question is, if it is possible, that through the size-exclusion the salt-concentration increased to such a high level, that this will damage the enzyme. Is it possible that the elution of NaCl is so strong delayed, that at the top of the column unexpected high concentration of NaCl occur? Thanks for your help! Jan Jan R. Kr?hling Pharmacist Institute of Pharmacology, Toxicology and Clinical Pharmacy Technical University of Brunswick Mendelssohnstrasse 1 D-38106 Braunschweig E-Mail: J-R.Kraehling@tu-braunschweig.de Tel. 05 31 / 391 - 56 23 Fax. 05 31 / 391 - 81 82 From allison from nospam.com Wed Dec 10 10:48:24 2008 From: allison from nospam.com (Allison) Date: Wed Dec 10 11:51:12 2008 Subject: PAGE gel sticked on both plates In-Reply-To: References: Message-ID: <493faa4e$0$5531$9a6e19ea@news.newshosting.com> Nicosnake wrote: > HI everyone, i've been having this problem whit my acrylamide gels: after > acrylamide is polymerized between the two glass plates it's really hard to > separate both plates and after a long effort i can take them appart only to > realize that the gel has sticked to BOTH plates, the gel is actually cut in > half. I treat surfaces of both plates, one with bind silane (so the > acrylamide sticks to this one) and the other with Sigmacote to prevent the > gel from sticking. But still the gel sticks to both surfaces and after i > separate the glases it's useless. Has anyonehad this kind of trouble?? ANy > suggestion is accepted. I clean my plates with Windex every time just before using them - works fine to prevent sticking! Allison From novalidaddress from nurfuerspam.de Wed Dec 10 17:15:40 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Dec 10 17:22:38 2008 Subject: Size-Exclusion References: Message-ID: Hi Jan. Actually, NaCl (as all small ions) should distribute evenly across the column when it has been equilibrated. Can you perform online conductivity monitoring? - You should be able to "see" the constant salt concentration in the solvent. Another issue might be that your protein becomes too concentrated and then precipitates and/or denatures. In that case, try to work at a higher dilution or -if appropriate- try to optimize salt concentration, pH or add eg urea (maybe also sucrose or maltose will do, try to use something that does not disturb your activity assay) in order to stabilize your enzyme. Best reagrds, Wolfgang On Dec 10, 11:50?am, "Jan R. Kr?hling" wrote: > Hello! > > We are trying to establish the sixe-exclusion chromatography in our lab to purify the enzyme of our interest. In the purification-steps before the ?size exclusion we observe that a NaCl-concentration of 1M stabilize the enzyme better than lower NaCl concentrations. So we tried the size exclusion at first with a high salt concentration, but the enzyme wasn't detectable. At lower concentrations the enzyme is detectable and active. My question is, if it is possible, that through the size-exclusion the salt-concentration increased to such a high level, that this will damage the enzyme. Is it possible that the elution of NaCl is so strong delayed, that at the top of the column unexpected high concentration of NaCl occur? > > Thanks for your help! > > Jan From ebs15242 from creighton.edu Wed Dec 10 18:06:37 2008 From: ebs15242 from creighton.edu (Ed Siefker) Date: Wed Dec 10 20:07:26 2008 Subject: Protein domain database? Message-ID: <49404B7D.6060502@creighton.edu> I am trying to answer what should be a simple question. That is, which residues of FGFR3 are intracellular? I'm shocked to find that this information isn't in any of the usual databases. NCBI's gene page for FGFR3 doesn't have it. If I click through to the protein sequence, it's not marked as a feature. The RCSB protein data bank doesn't seem to have this information either. Now obviously I can search the literature and find a reference that has this information. I'll be doing that tomorrow. But isn't this the kind of thing we should be able to find out with a few clicks in a database? Is there a database that has this kind of information, and I'm just not finding it? If not, why not? -Ed From kaj.stenberg from helsinki.fi.invalid Thu Dec 11 03:23:07 2008 From: kaj.stenberg from helsinki.fi.invalid (kaj.stenberg@helsinki.fi.invalid) Date: Thu Dec 11 12:08:58 2008 Subject: Protein domain database? References: Message-ID: Ed Siefker wrote: > I am trying to answer what should be a simple question. > That is, which residues of FGFR3 are intracellular? www.expasy.org search swiss-prot for FGFR3, look under "features" in the results. -- Kaj From cjtcdy from gmail.com Thu Dec 11 05:26:43 2008 From: cjtcdy from gmail.com (chiranjit chowdhury) Date: Thu Dec 11 12:09:06 2008 Subject: kind help please Message-ID: <2021705c0812110226k5af82868ub8a7c92b1d116738@mail.gmail.com> Can any body please tell me how peptidoglycan can be purified and isolated with the use of TLC or HPTLC from Escherichia coli? Regards -- Chiranjit Chowdhury Department of Biotechnology Indian Institute of Technology, Kharagpur Kharagpur, West Bengal, India Pin: 721302 Official email: chiranjit.chowdhury@iitkgp.ac.in From kumar from lifesensors.com Thu Dec 11 13:22:40 2008 From: kumar from lifesensors.com (Suresh Kumar) Date: Thu Dec 11 14:25:17 2008 Subject: Protein domain database? In-Reply-To: <200812111706.mBBH66V09227@net.bio.net> References: <200812111706.mBBH66V09227@net.bio.net> Message-ID: <899CFF650DAF1245801B4A694F0E8064574B6A@ls-sbs.ls.local> Hi Ed, Here is the information that you are looking for. http://www.uniprot.org/uniprot/P22607 Scroll down to the Sequence annotation section and you will find the answer. The sequence information is given as "potential" probably based on the prediction algorithm. You may still want to check the original reference to verify it. This information is also available through NCBI protein search. But you need to look harder. :) -Suresh -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of methods-request@oat.bio.indiana.edu Sent: Thursday, December 11, 2008 12:06 PM To: methods@magpie.bio.indiana.edu Subject: Methods Digest, Vol 43, Issue 9 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: Size-Exclusion (WS) 2. Protein domain database? (Ed Siefker) ---------------------------------------------------------------------- Message: 1 Date: Wed, 10 Dec 2008 14:15:40 -0800 (PST) From: WS Subject: Re: Size-Exclusion To: methods@net.bio.net Message-ID: Content-Type: text/plain; charset=ISO-8859-1 Hi Jan. Actually, NaCl (as all small ions) should distribute evenly across the column when it has been equilibrated. Can you perform online conductivity monitoring? - You should be able to "see" the constant salt concentration in the solvent. Another issue might be that your protein becomes too concentrated and then precipitates and/or denatures. In that case, try to work at a higher dilution or -if appropriate- try to optimize salt concentration, pH or add eg urea (maybe also sucrose or maltose will do, try to use something that does not disturb your activity assay) in order to stabilize your enzyme. Best reagrds, Wolfgang On Dec 10, 11:50?am, "Jan R. Kr?hling" wrote: > Hello! > > We are trying to establish the sixe-exclusion chromatography in our lab to purify the enzyme of our interest. In the purification-steps before the ?size exclusion we observe that a NaCl-concentration of 1M stabilize the enzyme better than lower NaCl concentrations. So we tried the size exclusion at first with a high salt concentration, but the enzyme wasn't detectable. At lower concentrations the enzyme is detectable and active. My question is, if it is possible, that through the size-exclusion the salt-concentration increased to such a high level, that this will damage the enzyme. Is it possible that the elution of NaCl is so strong delayed, that at the top of the column unexpected high concentration of NaCl occur? > > Thanks for your help! > > Jan ------------------------------ Message: 2 Date: Wed, 10 Dec 2008 17:06:37 -0600 From: Ed Siefker Subject: Protein domain database? To: methods@magpie.bio.indiana.edu Message-ID: <49404B7D.6060502@creighton.edu> Content-Type: text/plain; charset=ISO-8859-1; format=flowed I am trying to answer what should be a simple question. That is, which residues of FGFR3 are intracellular? I'm shocked to find that this information isn't in any of the usual databases. NCBI's gene page for FGFR3 doesn't have it. If I click through to the protein sequence, it's not marked as a feature. The RCSB protein data bank doesn't seem to have this information either. Now obviously I can search the literature and find a reference that has this information. I'll be doing that tomorrow. But isn't this the kind of thing we should be able to find out with a few clicks in a database? Is there a database that has this kind of information, and I'm just not finding it? If not, why not? -Ed ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 43, Issue 9 ************************************** From diegoromano76 from gmail.com Fri Dec 12 04:55:30 2008 From: diegoromano76 from gmail.com (diegoromano76@gmail.com) Date: Fri Dec 12 11:58:34 2008 Subject: pH and IPTG Message-ID: <0815b12e-b780-4841-8ff0-1be074cf9470@m12g2000vbp.googlegroups.com> Hi, I'm actually working on the expression of a protein in pET11b/pET24b- BL21(DE3)Star system: does anyone knows if there is an effect of the pH of the colture at the moment of induction on the IPTG uptake from the cells? diego From ebs15242 from creighton.edu Fri Dec 12 13:11:08 2008 From: ebs15242 from creighton.edu (Ed Siefker) Date: Fri Dec 12 16:32:14 2008 Subject: Protein domain database? In-Reply-To: <899CFF650DAF1245801B4A694F0E8064574B6A@ls-sbs.ls.local> References: <200812111706.mBBH66V09227@net.bio.net> <899CFF650DAF1245801B4A694F0E8064574B6A@ls-sbs.ls.local> Message-ID: <4942A93C.80906@creighton.edu> Thanks a ton. I knew that information had to be easily accessible somewhere. Now I know. -Ed Suresh Kumar wrote: > Hi Ed, > > Here is the information that you are looking for. > > http://www.uniprot.org/uniprot/P22607 > > Scroll down to the Sequence annotation section and you will find the answer. The sequence information is given as "potential" probably based on the prediction algorithm. > You may still want to check the original reference to verify it. > > This information is also available through NCBI protein search. > But you need to look harder. :) > > -Suresh > > From quangtranho from yahoo.com Mon Dec 15 11:08:34 2008 From: quangtranho from yahoo.com (TRAN HO QUANG) Date: Mon Dec 15 14:51:03 2008 Subject: How primer concentration can me measured? Message-ID: <62964.72340.qm@web30403.mail.mud.yahoo.com> Does anyone tell me how can measure primer concentration (working solution) I am facing problems: the PCR reactions are not consistent. It is suggested that primer concentrations are not exact as it is 10 uM (working solution). The questions is can i use agarose or NanoDrop to check primer concentration?. Any suggestions are warmly welcome! Digifellow From b.ganss from utoronto.ca Mon Dec 15 23:21:54 2008 From: b.ganss from utoronto.ca (Bernhard Ganss) Date: Tue Dec 16 10:06:48 2008 Subject: For all you, electroporation fans! (Was: Re: Electro-transformation of mammalian cells) Message-ID: <001801c95f35$d4553870$7cffa950$@ganss@utoronto.ca> Hi I am just testing if this email account still works - I have many questions re. mammalian electroporation and am trying to reach Dima Klenchin. Thanks ********************* Bernhard Ganss, Ph.D. Associate Professor CIHR Group in Matrix Dynamics University of Toronto, Faculty of Dentistry 150 College Street, room 234 Toronto, Ontario M5S 3E2, CANADA Phone: +1-416-978-8728 (office) +1-416-978-6624 (lab) Fax: +1-416-978-5956 email: b.ganss@utoronto.ca web: http://www.cihrmatrix.ca From fulvio.celsi from gmail.com Tue Dec 16 07:19:01 2008 From: fulvio.celsi from gmail.com (Fulvio Celsi) Date: Tue Dec 16 10:06:59 2008 Subject: How primer concentration can me measured? In-Reply-To: <62964.72340.qm@web30403.mail.mud.yahoo.com> References: <62964.72340.qm@web30403.mail.mud.yahoo.com> Message-ID: <49479CB5.1080705@gmail.com> I guess you can try using NanoDrop as it should measure very small quantity of primers..on agarose gel usually primers runs as dimers and this gives a very "fuzzy" band difficult to quantify good luck! (and..redissolving primers and making another working solution?? :D ) TRAN HO QUANG ha scritto: > Does anyone tell me how can measure primer concentration (working solution) > > I am facing problems: the PCR reactions are not consistent. It is suggested that primer concentrations are not exact as it is 10 uM (working solution). The questions is can i use agarose or NanoDrop to check primer concentration?. > > > > Any suggestions are warmly welcome! > > Digifellow > > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > -- Fulvio Celsi BsC,PhD Dept. of Experimental and Diagnostic Medicine Section of General Pathology University of Ferrara Via Borsari 46 44100 Ferrara Italy Mobile: +393286489131 http://salvarelaricerca.blogspot.com/ From fulvio.celsi from gmail.com Tue Dec 16 07:29:54 2008 From: fulvio.celsi from gmail.com (Fulvio Celsi) Date: Tue Dec 16 10:07:05 2008 Subject: Co-IP problems Message-ID: <49479F42.9030504@gmail.com> Dear All I'm looking for suggestions on CoImmunoPrecipitation.... The protein I'm interested in CoIP has a MW of 75Kd, so any time I made the experiment, on the CoIP lane I saw a smear from the Igg (around 60kd if I know well). How can I reduce this effect? I cannot buy another Ab (from a different species) thing that I suppose would help...can I cross-link ab to the protein-A beads? if yes...how?? Thanks a lot in advance Fulvio -- Fulvio Celsi BsC,PhD Dept. of Experimental and Diagnostic Medicine Section of General Pathology University of Ferrara Via Borsari 46 44100 Ferrara Italy Mobile: +393286489131 http://salvarelaricerca.blogspot.com/ From L.Kamalian from liverpool.ac.uk Tue Dec 16 08:17:15 2008 From: L.Kamalian from liverpool.ac.uk (Kamalian, Laleh) Date: Tue Dec 16 10:07:10 2008 Subject: How primer concentration can me measured? In-Reply-To: <62964.72340.qm@web30403.mail.mud.yahoo.com> References: <62964.72340.qm@web30403.mail.mud.yahoo.com> Message-ID: <31DC2DFFD70C174B8E3072803F894F9183C364@EVSSTAFF1.livad.liv.ac.uk> Hi. I usually use NanoDrop to double check the concentration written on the tube anyway. Only make sure you use the specific OD on the tube for the primer. Best of luck. L. -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of TRAN HO QUANG Sent: 15 December 2008 16:09 To: methods@magpie.bio.indiana.edu Subject: How primer concentration can me measured? Does anyone tell me how can measure primer concentration (working solution) I am facing problems: the PCR reactions are not consistent. It is suggested that primer concentrations are not exact as it is 10 uM (working solution). The questions is can i use agarose or NanoDrop to check primer concentration?. Any suggestions are warmly welcome! Digifellow _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From allison from nospam.com Tue Dec 16 10:48:20 2008 From: allison from nospam.com (Allison) Date: Tue Dec 16 13:39:04 2008 Subject: Co-IP problems In-Reply-To: References: Message-ID: <49479310$0$5559$9a6e19ea@news.newshosting.com> Fulvio Celsi wrote: > Dear All > I'm looking for suggestions on CoImmunoPrecipitation.... The protein > I'm interested in CoIP has a MW of 75Kd, so any time I made the > experiment, on the CoIP lane I saw a smear from the Igg (around 60kd if > I know well). How can I reduce this effect? I cannot buy another Ab > (from a different species) thing that I suppose would help...can I > cross-link ab to the protein-A beads? if yes...how?? > Thanks a lot in advance > Fulvio > Could you run a non-reducing gel? Then the IgG would run at 150K vs 75K for your protein (assuming the latter is not subunits as well). Allison From fulvio.celsi from gmail.com Tue Dec 16 13:53:57 2008 From: fulvio.celsi from gmail.com (Fulvio Celsi) Date: Tue Dec 16 18:33:50 2008 Subject: Co-IP problems In-Reply-To: <49479310$0$5559$9a6e19ea@news.newshosting.com> References: <49479310$0$5559$9a6e19ea@news.newshosting.com> Message-ID: <4947F945.3020409@gmail.com> Allison ha scritto: > Fulvio Celsi wrote: >> Dear All >> I'm looking for suggestions on CoImmunoPrecipitation.... The protein >> I'm interested in CoIP has a MW of 75Kd, so any time I made the >> experiment, on the CoIP lane I saw a smear from the Igg (around 60kd >> if I know well). How can I reduce this effect? I cannot buy another >> Ab (from a different species) thing that I suppose would help...can I >> cross-link ab to the protein-A beads? if yes...how?? >> Thanks a lot in advance >> Fulvio >> > > Could you run a non-reducing gel? Then the IgG would run at 150K vs > 75K for your protein (assuming the latter is not subunits as well). > Allison ehm..unfortunatly not...the whole protein is bigger (tetramer)...but good idea... thanks! Fulvio > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Fulvio Celsi BsC,PhD Dept. of Experimental and Diagnostic Medicine Section of General Pathology University of Ferrara Via Borsari 46 44100 Ferrara Italy Mobile: +393286489131 http://salvarelaricerca.blogspot.com/ From vasu.nanobiotech91 from gmail.com Wed Dec 17 03:18:06 2008 From: vasu.nanobiotech91 from gmail.com (Shri) Date: Wed Dec 17 10:59:23 2008 Subject: protocol for estimation for estimation of lignin from Sugarcane bagasse Message-ID: hi ...this is shrinivas doin PhD from India i want estimation of lignin from Sugarcane bagasse..protocol .. or else send me the TAPPI standard protocol.. which could help me estmate lignin conc. present in the sugarcane bagasse.. with regards D.shrinivas From editor from gene-quantification.info Wed Dec 17 04:03:08 2008 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Wed Dec 17 10:59:59 2008 Subject: qPCR NEWS December 2008 Message-ID: qPCR NEWS December 2008 ---------------------------------------------------------------------------= ----- Dear researcher, dear Gene Quantification page reader, Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene Quantification homepage. The focus of this newsletter issue is: - Final Call for TALKS & POSTERS for qPCR 2009 Event =3D> http://www.qPCR= 2009.net - European wide qPCR application workshops =3D> register now ! - REST 2008 update - PCR efficiency page updated with the newest papers in the field - Online translation service of the Gene Quantification =3D> http://translation.gene-quantification.info/ ---------------------------------------------------------------------------= ----- Dear qPCR NEWS-Reader, Best Wishes for the Holiday Season and a Happy and Successful New Year 2009 ! ---------------------------------------------------------------------------= ----- REST 2008 update REST 2008 builds on its predecessor REST 2005 with significant improvements to randomization algorithms. This new revision introduces alternative data inputs such as single run efficiency and amplification take-off point, alleviating the need to set amplification plot thresholds. REST 2008 is a standalone software package for analyzing gene expression using real-time amplification data. The software addresses issues surrounding the measurement of uncertainty in expression ratios by introducing randomization and bootstrapping techniques. New confidence intervals for expression levels also allow measurement of not only the statistical significance of deviations but also their likely magnitude, even in the presence of outliers. Whisker box plots provide a visual representation of variation for each gene, highlighting potential issues such as distribution skew. REST 2008 builds on its predecessor REST 2005 with significant improvements to randomization algorithms. This new revision introduces alternative data inputs such as single sample efficiency and amplification take- off point, alleviating the need to set amplification plot thresholds. http://rest.gene-quantification.info/ What's NEW since REST 2005 ? NEW - REST-RG mode A new method of input has been introduced, allowing users to copy and paste results from the Rotor-Gene software's Comparative Quantitation analysis. This is an alternative to importing standard curve and CT results. See REST-RG Mode chapter for more details. NEW - Whisker-Box Plots Exportable Whisker-Box plots can now be exported by right-clicking on the graph. NEW - Improved randomisation Improvements to the randomisation algorithms have been made, making confidence intervals much tighter, and p-values more accurate. In previous versions the pair-wise fixed reallocation was incorrectly matching the gene of interest CT with the incorrect reference CT, this issue has been rectified in REST 2008. NEW - Handling of standard curve variation REST 2008 no longer takes into account the variation of the standard curve and implements improvements to the calculation of confidence intervals and p-values. In previous versions, the software would randomly pick two points from the standard curve and calculate an efficiency based on that. However there is a situation when two points are chosen that lie close to each other on the standard curve, this can cause a bogus efficiency which adds unnecessary outliers to the random distribution. We now calculate the efficiency by determining the line of best fit for the standard curve, this efficiency is used through the randomisation process. Download =3D> REST 2008 software http://www.gene-quantification.de/download.html#rest-2008 Download =3D> Manual REST 2008 version 2.0.7 http://www.gene-quantification.de/REST2008_Manual_v207.pdf Slide Show REST-2008 http://www.gene-quantification.de/rest-2008.html#slides The new stand-alone software versions REST 2008 was programmed and designed by Matthew Herrmann, David Chiew, and Birgit Speller working at Corbett Research (Sydney, Australia) and Michael W. Pfaffl, Technical University of Munich, Germany. ---------------------------------------------------------------------------= ----- update in determination of real-time PCR amplification efficiency taken from Chapter 3: Quantification strategies in real-time PCR in: A-Z of quantitative PCR ( Editor: S.A. Bustin ) International University Line (IUL), La Jolla, CA, USA Individual samples generate different and individual fluorescence histories in kinetic RT-PCR. The shapes of amplification curves differ in the steepness of any fluorescence increase and in the absolute fluorescence levels at plateau depending on background fluorescence levels. The PCR efficiency has a major impact on the fluorescence history and the accuracy of the calculated expression result and is critically influenced by PCR reaction components. Efficiency evaluation is an essential marker in gene quantification procedure. Constant amplification efficiency in all compared samples is one important criterion for reliable comparison between samples. This becomes crucially important when analyzing the relationship between an unknown sequence versus a standard sequence, which is performed in all relative quantification models. In experimental designs employing standardization with housekeeping genes, the demand for invariable amplification efficiency between target and standard is often ignored, despite the fact that corrections have been suggested. A correction for efficiency, as performed in efficiency corrected mathematically models, is strongly recommended and results in a more reliable estimation of the =91real expression ratio=92 compared to NO efficiency correction. Small efficiency differences between target and reference gene generate false expression ratio, and the researcher over- or under-estimates the =91real=92 initial mRNA amount. The assessment of the exact amplification efficiencies of target and reference genes must be carried out before any calculation of the normalized gene expression is done. LightCycler Relative Expression Software, Q-Gene, REST and REST-XL software applications allow the evaluation of amplification efficiency plots. Different tissues exhibit different PCR efficiencies, caused by RT inhibitors, PCR inhibitors and by variations in the total RNA fraction pattern extracted. Find lots of useful papers here =3D> http://efficiency.gene-quantification.= info/ latest papers published in 2008: - A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR (Rutledge & Stewart, 2008) - A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition (Guescini et al., 2008) - Highly accurate sigmoidal fitting of real-time PCR data by introducing a parameter for asymmetry. (Spiess et al., 2008) - qPCR: an R package for sigmoidal model selection in quantitative real-time polymerase chain reaction analysis (Ritz & Spiess, 2008) - Critical evaluation of methods used to determine amplification efficiency refutes the exponential character of real-time PCR (Rutledge & Stewart 2008) - A new method for robust quantitative and qualitative analysis of real-time PCR (Shain & Clemens, 2008) - CAmpER - Real-time PCR analysis software - Calculation of Amplification Efficiencies for RT-PCR experiments is a tool for the automatic analysis of real time PCR experiments. Automatic analysis, annotation and storage of real-time PCR experiments performed with different qPCR systems, currently the LightCycler 2, LC480, RotorGene and the Opticon. ---------------------------------------------------------------------------= ----- online translation Since October 2008 we provide an online translation service of the Gene Quantification pages to several languages. Please recognize this is an automatic and robotic based translation service, and therefore we can give NO guarantee about the generated content. It should help to understand the "rough" content of the Gene Quantification pages, but still the original is the ENGLISH version: http://translation.gene-quantification.info/ http://www.gene-quantification.asia http://chinese.gene-quantification.asia/ http://dutch.gene-quantification.info/ http://french.gene-quantification.info/ http://german.gene-quantification.info/ http://greek.gene-quantification.info/ http://italian.gene-quantification.info/ http://japanese.gene-quantification.asia/ http://korean.gene-quantification.asia/ http://portuguese.gene-quantification.info/ http://russian.gene-quantification.asia/ http://spanish.gene-quantification.info/ ---------------------------------------------------------------------------= ----- With the new qPCR INFO PORTAL and all the presented tools we will help you with to find the right information about qPCR and related topics in Molecular Biology in the literature and in the World Wide Web. =3D> Papers / Protocols / Methods / Databases / Alets / Feeds / Books / Forums / E-mail / Directory http://infoportal.gene-quantification.info/ ---------------------------------------------------------------------------= ----- Upcoming Events World-wide academic and commercial qPCR Events http://events.gene-quantification.info/ Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars, qPCR Education Program, etc. Please submit your qPCR event here =3D> events@gene- quantification.info ---------------------------------------------------------------------------= ----- qPCR 2009 EVENT - 9 - 13 March 2009 more news here =3D> http://www.qPCR2009.net download event FLYER =3D> http://www.bioeps.com/qpcr2009/qPCR-2009-3rd-an= nouncement.pdf It is a pleasure to announce the Nobel Prize Laureate Kary Mullis at the qPCR 2009 event in an own session =9325th Anniversary of PCR=94 List of confirmed speakers =3D> http://speakers.qpcr2009.net/ An industrial exhibition with the 30 world leading companies will be held during the qPCR Symposium March 9-11 =3D> http://exhibition.qpcr2009= .net/ Our sponsors =3D> http://sponsors.qpcr2009.net/ Please register until 31st December to get the early registartion fee =3D> http://registration.qpcr2009.net/ Keynote lectures The Pioneer in PCR Kary B. Mullis 1993 Nobel Prize in Chemistry - 25th Anniversary of PCR Stephen A. Bustin Professor of Molecular Science, Institute of Cell and Molecular Science, Queen Mary's School of Medicine and Dentistry, University of London, UK - A new qPCR assay for the detection of Clostridium difficile - MIQE- guidelines for publication of qPCR data Ken Livak Senior Scientific Fellow, Fluidigm Corporation,San Francisco, CA, US - Moving from qPCR Assays to qPCR Arrays. ---------------------------------------------------------------------------= ----- qPCR WORKSHOP BioEPS GmbH / TATAA Biocenter Germany - qPCR Application workshops At the TATAA Biocenter Germany we offer qPCR application workshops, a 3-day qPCR Core Module and a 2-day qPCR Biostatistics Module. All courses are held regularly in G=F6teborg, Sweden, in English and in Freising-Weihenstephan, Germany, in German and English, and in Prague, Czech Republic in English and Czech. Depending on the occasion the workshop language and the different prices may apply. Further customized workshops and specialized trainings will be held as well across Europe and world-wide. TATAA Biocenter Germany workshops are held in cooperation with BioEPS GmbH, located at the campus of the Technical University of Munich, in Freising-Weihenstephan, very close to the Munich Airport (MUC). For more information and registration, please see our web page: =3D> http://TATAA.gene-quantification.info/ Course Occasions 2008/2009: 3-day qPCR Core Module (Mon. - Wed.) 2-day BioStatistics Module (Thu. - Fri.) single-cell qPCR Module (Mon. - Wed.) 26 - 30th January 2009 (E) in Freising, Germany, English language 26 - 30th January 2009 (E) in Freising, Germany, English language 16 - 18 February 2009 (E) in Freising, NEW microRNA & qPCR 12 - 13 March 2009 (E) various 2-day courses at the qPCR 2009 Event =3D> http://workshops.qpcr2009.net/ ---------------------------------------------------------------------------= ----- Forward Please send the qPCR NEWS to further scientists and friends who are interested in qPCR ! Best regards, Michael W. Pfaffl responsible Editor of the Gene Quantification Pages http://www.gene-quantification.info ---------------------------------------------------------------------------= ----- If this newsletter is not displayed correctly by your email client, please use following link: http://qpcrnews.gene-quantification.info/ The qPCR NEWS and the Gene Quantification Pages are educational sites with the only purpose of facilitating access to qPCR related information on the internet. The qPCR NEWS and the Gene Quantification Pages are edited by Michael W. Pfaffl. Copyright =A9 2005 - 2008 All rights reserved. Any unauthorized use, reproduction, or transfer of this message or its contents, in any medium, is strictly prohibited. Disclaimer & Copyrights are displayed on the homepage www.gene-quantification.com The qPCR newsletter was end to [alle-adressen] To subscribe or change your e-mail address in qPCR NEWS, and if you would like to receive future issues FREE of charge, please send an e- mail with the subject SUBSCRIBE to mailto:newsletter@gene- quantification.info?subject=3DSUBSCRIBE From jgardn from lsuhsc.edu Wed Dec 17 14:51:00 2008 From: jgardn from lsuhsc.edu (Gardner, Jason D.) Date: Wed Dec 17 18:48:19 2008 Subject: lysyl oxidase activity Message-ID: <9DE950516C27AB4FBD3DCECDC802AE89DDA695@EXCHBE2.master.lsuhsc.edu> We are currently using the Amplex Red method to evaluate lysyl oxidase enzymatic activity in cell-conditioned media and it is working very well. We would like to perform the same assay in tissue extracts. Our first attempts have produced mixed results and we are currently tweaking multiple variables (Amplex red, substrate, temp, protein levels, etc.) to improve upon the method. Is anyone out there measuring lysyl oxidase activity in tissue? Regards. From shahrzadjalali from gmail.com Fri Dec 19 09:55:47 2008 From: shahrzadjalali from gmail.com (Shahrzad Jalali) Date: Fri Dec 19 10:36:41 2008 Subject: An inquiry Message-ID: To whom it may concern, I am from U of Toronto and have recently joined to your group. Had some problems in dissolving a compound and was wondering if I can get any suggestion from the expert people. I have to do i.p. injections to the rats and the compound of interest is src kinase inhibitor PP1. http://www.biomol.com/SiteData/docs/productdata/ei275.pdf. As I got the information from the literature, it has been mentioned in several papers that this material is dissolved in DMSO and further diluted in the PBS prior to the injection to rats. In the data sheet of the company it is also mentioned that this material should be dissolved in DMSO (25 mg/ml). Each vial had 5mg in it, I initially added 200ul of DMSO to that and completely dissolve it by pipetting up and down, but when I added PBS (as indicated in the papers), all the compound was precipitated and I got just a colloidal solution. The company did not have any suggestion, as they told that it is hydrophobic material. I don't know how the papers have done that. On the other hand I have to dilute it in PBS because injecting DMSO to the rats has other effects which can interfere with my experimental plan. I should mention that heating the compound up to 37C for several hours and its repetitive agitation did not help this compound go to the solution. I would appreciate if anybody can give suggestion in this regard and urgently waiting for the nswer, Thanks, Shahrzad From hroychow from nmsu.edu Fri Dec 19 12:29:33 2008 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Fri Dec 19 14:30:48 2008 Subject: An inquiry In-Reply-To: References: Message-ID: <1240.71.210.225.55.1229707773.squirrel@webmail.nmsu.edu> I don't believe you can expect a solution of PP1 in PBS. It will form a colloidal suspension. A colloidal suspension will also elicit immune response. Remember that the peritonial fluid is also polar and the PP1 will come out of solution there too. DMSO, however, does the rat very little damage in microliter quantities intra-peritonially. I believe the PBS is used to minimize any shock. PP1 is soluble also in ethanol, and it may be a better alternative solvent for the injection. Just a thought! > To whom it may concern, > > I am from U of Toronto and have recently joined to your group. Had some > problems in dissolving a compound and was wondering if I can get any > suggestion from the expert people. > > I have to do i.p. injections to the rats and the compound of interest is > src > kinase inhibitor PP1. > http://www.biomol.com/SiteData/docs/productdata/ei275.pdf. As I got the > information from the literature, it has been mentioned in several papers > that this material is dissolved in DMSO and further diluted in the PBS > prior > to the injection to rats. > In the data sheet of the company it is also mentioned that this material > should be dissolved in DMSO (25 mg/ml). Each vial had 5mg in it, I > initially added 200ul of DMSO to that and completely dissolve it by > pipetting up and down, but when I added PBS (as indicated in the papers), > all the compound was precipitated and I got just a colloidal solution. The > company did not have any suggestion, as they told that it is hydrophobic > material. I don't know how the papers have done that. On the other hand I > have to dilute it in PBS because injecting DMSO to the rats has other > effects which can interfere with my experimental plan. I should mention > that > heating the compound up to 37C for several hours and its repetitive > agitation did not help this compound go to the solution. > > I would appreciate if anybody can give suggestion in this regard and > urgently waiting for the nswer, > Thanks, > Shahrzad > _______________________________________________ > 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 SBrown from ccia.unsw.edu.au Fri Dec 19 12:33:48 2008 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Fri Dec 19 14:30:56 2008 Subject: methylcellulose colony question Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A02972BC5@mail01.ccia.local> Hi, I have been growing Nalm-6 colonies in methocult for about a year now, the ratio of media is 1.6ml methocult, 1.2ml IMDM and 1.2ml FCS with or without drug depending on what i am doing. Has anyone had any experience with maintaining the methocult for at least 3 weeks, frequently my methocult looks as though is has dessicated, i have water in the incubator and i also plate 2 wells with PBS to ensure humidity...but...it still happens, any ideas or suggestions? S Scott Brown PhD Candidate Children's Cancer Institute Australia for Medical Research is the only independent medical research institute in Australia devoted to research into the causes, prevention, better treatment and ultimately a cure of childhood cancer. Our vision is to save the lives of all children with cancer and to eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error please contact the sender immediately and destroy the original message. From SBrown from ccia.unsw.edu.au Fri Dec 19 12:38:40 2008 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Fri Dec 19 14:31:03 2008 Subject: RACE sequencing problem Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A02972BC6@mail01.ccia.local> Hi, I have been sequencing constructs about 1kb in size that i have obtained from a second race reaction, i have pruified the product, spec'd it and ensured when run on a gel i get a band. This has worked for a while, however the last 1 or so constructs i have had sequenced have failed, the chromatogram looks as though the primer has not even bound to the template, it is simply noise that drops off to an almost undetectable signal. Can anyone make any suggestions as to what I should be looking at to troubleshoot this? I sent my positive control as well as 1 sample from my next set of rave reactions to be sequenced, both were successful, then when i sent the remaining 8, all 8 failed. Might i need to redesign my primer i am using for sequencing? Any other thoughts? S Scott Brown PhD Candidate Children's Cancer Institute Australia for Medical Research is the only independent medical research institute in Australia devoted to research into the causes, prevention, better treatment and ultimately a cure of childhood cancer. Our vision is to save the lives of all children with cancer and to eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error please contact the sender immediately and destroy the original message. From jayanta.tcbckv from gmail.com Fri Dec 19 12:53:03 2008 From: jayanta.tcbckv from gmail.com (Jayanta Tarafdar) Date: Fri Dec 19 14:31:08 2008 Subject: RNAi Message-ID: Dear Subscribers Methods Digest We have been working on the backcross breeding of rice with transgenic rice carrying RNAi gene against virus. Would anybody help me regarding use of non-radioactive probe for analysis of the transcript. I would appreciate if anybody can give short-cut method in this regard and urgently waiting for the answer. Jayanta India From stxsz1 from nottingham.ac.uk Sun Dec 21 06:11:48 2008 From: stxsz1 from nottingham.ac.uk (Zhong Silin) Date: Sun Dec 21 14:31:41 2008 Subject: a follow up question regarding dissolving H2O insoluable compounds in DMSO References: <200812201703.mBKH3FV27782@net.bio.net> Message-ID: <5D7E3A8B871FD645A3A9FB96F3951EF8433220@VUIEXCHA.ad.nottingham.ac.uk> Hi I saw the interesting discussion of dissolving PP1 in PBS. I always take it for granted that one can dissolve water insoluble stuff in an organic solvent then add it to a water based solution. But I have never thought about it. Why is that possible? Why it wont precipitate out from the solution? That should still depends on the compound's solubility in water. Does it mean the organic solution is simply for a high concentration stock? BTW, is there a database for drug solubility? have a nice Xmas SZ ________________________________ From: methods-bounces@oat.bio.indiana.edu on behalf of methods-request@oat.bio.indiana.edu Sent: Sat 20/12/2008 17:03 To: methods@magpie.bio.indiana.edu Subject: Methods Digest, Vol 43, Issue 16 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: An inquiry (Dr. Hiranya S. Roychowdhury) 2. RNAi (Jayanta Tarafdar) 3. methylcellulose colony question (Scott Brown) 4. RACE sequencing problem (Scott Brown) ---------------------------------------------------------------------- Message: 1 Date: Fri, 19 Dec 2008 10:29:33 -0700 (MST) From: "Dr. Hiranya S. Roychowdhury" Subject: Re: An inquiry To: "Shahrzad Jalali" Cc: methods@magpie.bio.indiana.edu Message-ID: <1240.71.210.225.55.1229707773.squirrel@webmail.nmsu.edu> Content-Type: text/plain;charset=iso-8859-1 I don't believe you can expect a solution of PP1 in PBS. It will form a colloidal suspension. A colloidal suspension will also elicit immune response. Remember that the peritonial fluid is also polar and the PP1 will come out of solution there too. DMSO, however, does the rat very little damage in microliter quantities intra-peritonially. I believe the PBS is used to minimize any shock. PP1 is soluble also in ethanol, and it may be a better alternative solvent for the injection. Just a thought! > To whom it may concern, > > I am from U of Toronto and have recently joined to your group. Had some > problems in dissolving a compound and was wondering if I can get any > suggestion from the expert people. > > I have to do i.p. injections to the rats and the compound of interest is > src > kinase inhibitor PP1. > http://www.biomol.com/SiteData/docs/productdata/ei275.pdf. As I got the > information from the literature, it has been mentioned in several papers > that this material is dissolved in DMSO and further diluted in the PBS > prior > to the injection to rats. > In the data sheet of the company it is also mentioned that this material > should be dissolved in DMSO (25 mg/ml). Each vial had 5mg in it, I > initially added 200ul of DMSO to that and completely dissolve it by > pipetting up and down, but when I added PBS (as indicated in the papers), > all the compound was precipitated and I got just a colloidal solution. The > company did not have any suggestion, as they told that it is hydrophobic > material. I don't know how the papers have done that. On the other hand I > have to dilute it in PBS because injecting DMSO to the rats has other > effects which can interfere with my experimental plan. I should mention > that > heating the compound up to 37C for several hours and its repetitive > agitation did not help this compound go to the solution. > > I would appreciate if anybody can give suggestion in this regard and > urgently waiting for the nswer, > Thanks, > Shahrzad > _______________________________________________ > 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 ------------------------------ Message: 2 Date: Fri, 19 Dec 2008 23:23:03 +0530 From: "Jayanta Tarafdar" Subject: RNAi To: methods@magpie.bio.indiana.edu Message-ID: Content-Type: text/plain; charset=ISO-8859-1 Dear Subscribers Methods Digest We have been working on the backcross breeding of rice with transgenic rice carrying RNAi gene against virus. Would anybody help me regarding use of non-radioactive probe for analysis of the transcript. I would appreciate if anybody can give short-cut method in this regard and urgently waiting for the answer. Jayanta India ------------------------------ Message: 3 Date: Sat, 20 Dec 2008 04:33:48 +1100 From: "Scott Brown" Subject: methylcellulose colony question To: Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A02972BC5@mail01.ccia.local> Content-Type: text/plain; charset="iso-8859-1" Hi, I have been growing Nalm-6 colonies in methocult for about a year now, the ratio of media is 1.6ml methocult, 1.2ml IMDM and 1.2ml FCS with or without drug depending on what i am doing. Has anyone had any experience with maintaining the methocult for at least 3 weeks, frequently my methocult looks as though is has dessicated, i have water in the incubator and i also plate 2 wells with PBS to ensure humidity...but...it still happens, any ideas or suggestions? S Scott Brown PhD Candidate Children's Cancer Institute Australia for Medical Research is the only independent medical research institute in Australia devoted to research into the causes, prevention, better treatment and ultimately a cure of childhood cancer. Our vision is to save the lives of all children with cancer and to eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error please contact the sender immediately and destroy the original message. ------------------------------ Message: 4 Date: Sat, 20 Dec 2008 04:38:40 +1100 From: "Scott Brown" Subject: RACE sequencing problem To: Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A02972BC6@mail01.ccia.local> Content-Type: text/plain; charset="iso-8859-1" Hi, I have been sequencing constructs about 1kb in size that i have obtained from a second race reaction, i have pruified the product, spec'd it and ensured when run on a gel i get a band. This has worked for a while, however the last 1 or so constructs i have had sequenced have failed, the chromatogram looks as though the primer has not even bound to the template, it is simply noise that drops off to an almost undetectable signal. Can anyone make any suggestions as to what I should be looking at to troubleshoot this? I sent my positive control as well as 1 sample from my next set of rave reactions to be sequenced, both were successful, then when i sent the remaining 8, all 8 failed. Might i need to redesign my primer i am using for sequencing? Any other thoughts? S Scott Brown PhD Candidate Children's Cancer Institute Australia for Medical Research is the only independent medical research institute in Australia devoted to research into the causes, prevention, better treatment and ultimately a cure of childhood cancer. Our vision is to save the lives of all children with cancer and to eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error please contact the sender immediately and destroy the original message. ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 43, Issue 16 *************************************** 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 vasu.nanobiotech91 from gmail.com Thu Dec 25 12:15:31 2008 From: vasu.nanobiotech91 from gmail.com (Shri) Date: Thu Dec 25 16:01:00 2008 Subject: Gewtting the concentration of PCR product and cloning Message-ID: Hi all members, I have amplified PCR product of 1250 bp from Genomic DNA my question is 1 Whether it can be cloned in TA cloning vector the required size. 2. how to calculate the concentration of the PCR product after the reaction From shifalich from rediffmail.com Fri Dec 26 09:38:41 2008 From: shifalich from rediffmail.com (shifali chatrath) Date: Fri Dec 26 13:14:37 2008 Subject: Gewtting the concentration of PCR product and cloning Message-ID: <20081226143841.40625.qmail@f4mail205.rediffmail.com> ?Hi! I want to correct DK's point. If it is proofreading polymerase, 3'A will get depleted off as a result of proof reading. Taq Pol/ Long Pol amplified products can generally be used for TA cloning. Their fact will have that info. Secondly, to me the suggestion to Point 2 is tedious. Gel purified PCR product can be directly quantified and ligation reaction can be set up, depending upon the size of the insert, one needs to clone. 5:1::insert:vector is the optimal molar ratio for cloning insert of 1250bp. Just quantify the insert and go ahead. All the best. Shifali On Thu, 25 Dec 2008 Shri wrote : >Hi all members, >I have amplified PCR product of 1250 bp from Genomic DNA > my question is >1 Whether it can be cloned in TA cloning vector the required size. >2. how to calculate the concentration of the PCR product after the >reaction >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From aawara from pontiff-playground.org Fri Dec 26 17:17:21 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Fri Dec 26 19:15:01 2008 Subject: Gewtting the concentration of PCR product and cloning References: Message-ID: In , shifali chatrath wrote: > ?Hi! > I want to correct DK's point. If it is proofreading polymerase, 3'A will get depleted off as a result of proof reading. Taq Pol/ Long Pol amplified products can generally be used for TA cloning. Their fact will have that info. How are you correcting what Dima posted? You're simply repeating what he wrote (which was correct to begin with - PCR products amplified with a proof-reading polymerase must be A-tailed to be TA-cloned). > Secondly, to me the suggestion to Point 2 is tedious. Why is it tedious? Dima gave two methods to quantify PCR products - a) estimate their concentration by comparing band intensity to a marker of known concentration, or b) obtain a A260/A280 ratio by spectrophotometry. You've provided no method - other than the vacuous statement "Gel purified PCR product can be directly quantified ....". > Gel purified PCR product can be directly quantified and ligation reaction can be set up, depending upon the size of the insert, one needs to clone. 5:1::insert:vector is the optimal molar ratio for cloning insert of 1250bp. Just quantify the insert and go ahead. > All the best. > > Shifali Chatrath > Graduate Student > Protein science Lab > Dept. of Biological sciences > National University of Singapore > Singapore > +65-65161210 > +65-96393449 -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From shifalich from rediffmail.com Fri Dec 26 21:16:30 2008 From: shifalich from rediffmail.com (shifali chatrath) Date: Sat Dec 27 15:00:40 2008 Subject: Gewtting the concentration of PCR product and cloning Message-ID: <20081227021630.9801.qmail@f4mail-235-239.rediffmail.com> ? Dear all! Please read carefully what DK wrote before pointing fingers at me! Mr. Aawara is also repeating the same thing.[PCR products amplified with a proof-reading polymerase must be A-tailed ]. Please note that, if the enzyme has proof reading activity, it will remove 3'A and TA cloning cannot be done. And, it is Taq's property to put an extra A at 3' which has nothing to do with its proof reading. Also, If one has to do A260/A280, one will still be using spectrophotometer and quantifying A260,then why to run standards etc. P.S.: Dear Vasu! You can quantitate minute amounts using Nanodrop, you will need at least one microlitre to do that. If you dont have access to nanodrop, then probably, you need to follow the SIMPLE AGAROSE GEL AS SUGGESTED BY DK! HOpe fully it helps to you. P.S.: DK's supporters! This is an open scientific forum and I suppose evrybody has right to give their suggestions as easiest as possible and correct if one thinks that what is written is not what it is! still you can correct me if I wrote anything wrong about proofreading. I am sure, what I wrote is correct. Don't just bash at someone, as you all are trying to do. I did not rewrite what DK wrote. Rather, the time that you all spent on writing to me, better spend on reading carefully opinions of both of us. FYI: Please read , why after PCR, using Taq Pol, Long Pol, Advantage Pol etc. one needs to keep the PCR reaction at -20 quickly? You will know the difference between what I and DK wrote. Also, regarding ligation, refer to Promega's Rapid Ligation buffer literature (just a paper,available with Promega), they have tested variuos Insert: vector ratio, depending upon insert size , they have got different recombination efficiencies. This ratio was what I told additionally, and not just repeating DK's point. Hopefully, I made myself clear! Next time, better check your "VACUOUS STATEMENTS" ! Sorry to others out of all this! I know no body is that jobless to poke their noses into this matter. Shifali On Sat, 27 Dec 2008 Aawara Chowdhury wrote : >In , > shifali chatrath wrote: > > > ?Hi! > > I want to correct DK's point. If it is proofreading polymerase, 3'A will get depleted off as a result of proof reading. Taq Pol/ Long Pol amplified products can generally be used for TA cloning. Their fact will have that info. > >How are you correcting what Dima posted? You're simply repeating >what he wrote (which was correct to begin with - PCR products amplified >with a proof-reading polymerase must be A-tailed to be TA-cloned). > > > Secondly, to me the suggestion to Point 2 is tedious. > >Why is it tedious? Dima gave two methods to quantify PCR products - a) >estimate their concentration by comparing band intensity to a marker of >known concentration, or b) obtain a A260/A280 ratio by spectrophotometry. > >You've provided no method - other than the vacuous statement "Gel purified >PCR product can be directly quantified ....". > > > Gel purified PCR product can be directly quantified and ligation reaction can be set up, depending upon the size of the insert, one needs to clone. 5:1::insert:vector is the optimal molar ratio for cloning insert of 1250bp. Just quantify the insert and go ahead. > > All the best. > > > > Shifali Chatrath > > Graduate Student > > Protein science Lab > > Dept. of Biological sciences > > National University of Singapore > > Singapore > > +65-65161210 > > +65-96393449 > >-- >Email: echo 36434455860060025978157675027927670979097959886449930P | dc >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From shifalich from rediffmail.com Sun Dec 28 00:34:15 2008 From: shifalich from rediffmail.com (shifali chatrath) Date: Sun Dec 28 01:37:43 2008 Subject: Gewtting the concentration of PCR product and cloning Message-ID: <20081228053415.44232.qmail@f4mail211.rediffmail.com> P.S.: D.K. ?Why are you just beating about the bush? I had mentioned that I told optimal vector: insert ratio additionally, as your supporters found nothing new in what I said. And please dont explain to me all that mumbo jumbo of ligation etc. If it doesn't help you, just don't follow it. It better to start with something that you know than just making a wild guess. Secondly, if you are really interested on knowing about Avantage and long Pol, just give a google search. Now, Reitaliating what you said: If you used proofreading polymerase, it would have to be A-tailed first. If Taq and the like, no need to. These days, enzyme mix come with proof reading enzymes in them. therefore, just after PCR,if you want to continue just gel purify the product or if, to be used later, it should be quickly kept in -20, so that proof reading enzyme in that will polish away 3'A left by Taq. I hope I made myself clear now. Shifali On Sun, 28 Dec 2008 DK wrote : >In article , shifali chatrath wrote: > >FYI: Please read, why after PCR, using Taq Pol, Long Pol, Advantage Pol > >etc. one needs to keep the PCR reaction at -20 quickly? > >I don't know what Long Pol and Advantage Pol are but as far as >Taq goes, the answer is clear: no good reason at all! > > >Also, regarding ligation, refer to Promega's Rapid Ligation buffer literature, > >they have tested variuos Insert: vector ratio, depending upon insert size, > >they have got different recombination efficiencies. This ratio was what > >I told additionally. > >Just FYI: there is no such thing as optimal vector:insert ratio that >is applicable in all cases. It all depends on the particular case >(what is being cloned and how) and ultimate goal (e.g., vector >self-ligation background important or not, absolute number of >positive clones important or not, etc). > >Here is a simple question, see if you can figure it out: >Ligation 1: Vector only, no insert = 300 colonies (obviously, >all negative). >Ligation 2: Vector:insert = 1:4 molar ratio = 400 colonies >(1 in 6 positive) >Ligation 3: Vector:insert = 1:50 molar ratio = 70 colonies >(6 in 6 positive). > >Can you explain what's going on? And what would you say >is an optimal vector:insert ratio? > >DK >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From aawara from pontiff-playground.org Sun Dec 28 00:31:50 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Sun Dec 28 01:37:53 2008 Subject: Gewtting the concentration of PCR product and cloning References: Message-ID: In , shifali chatrath wrote: > ? > > Dear all! > Please read carefully what DK wrote before pointing fingers at me! > Mr. Aawara is also repeating the same thing.[PCR products amplified > with a proof-reading polymerase must be A-tailed ]. Please note that, if the enzyme has proof reading activity, it will remove 3'A and TA cloning cannot be done. And, it is Taq's property to put an extra A at 3' which has nothing to do with its proof reading. The problem is with your comprehension of English. Let me clarify: If one wrote, "PCR products .... ARE A-tailed", then it would indicate that products made with a proof-reading polymerase are already A-tailed. On the other hand, if one writes "PCR products .... MUST BE A-tailed", then it indicates that the products made with a proof-reading polymerase are NOT A-tailed, and therefore MUST BE A-tailed for them to be cloned into a TA-vector. Hope that helps you ..... AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From eenigoy from gmail.com Sun Dec 28 08:19:39 2008 From: eenigoy from gmail.com (yoginee budhkar) Date: Sun Dec 28 15:01:31 2008 Subject: Gewtting the concentration of PCR product and cloning Message-ID: <77ddf6c70812280519o38d15fe3xa925d70b8094847a@mail.gmail.com> Hello Shifali, Your first question is answered correctly already, for the quatification, read on... OD of 1 at 260nm corresponds to 50 ?g/ml for double-stranded DNA. Simply take absorbance of your PCR product on a nanophotometer/ a nanodrop. Or dilute it suitably to 1 ml and take A260 in a quartz cuvette, in any spectrophotometer. Multiply it with the dilution factor to find out the concentration of you PCR product. --Yg > On Thu, 25 Dec 2008 Shri wrote : > >Hi all members, > >I have amplified PCR product of 1250 bp from Genomic DNA > > my question is > >1 Whether it can be cloned in TA cloning vector the required size. > >2. how to calculate the concentration of the PCR product after the > >reaction > >_______________________________________________ > >Methods mailing list > >Methods@net.bio.net > >http://www.bio.net/biomail/listinfo/methods > > > Shifali Chatrath > Graduate Student > Protein science Lab > Dept. of Biological sciences > National University of Singapore > Singapore > +65-65161210 > +65-96393449 > > ------------------------------ > > From pow.joshi from gmail.com Sun Dec 28 15:38:29 2008 From: pow.joshi from gmail.com (Pow Joshi) Date: Sun Dec 28 16:31:53 2008 Subject: Fwd: Gewtting the concentration of PCR product and cloning In-Reply-To: <710764ea0812281200u4182658x1099339d1f3eeed0@mail.gmail.com> References: <710764ea0812281155u17dcd31l1364196ab1a941fe@mail.gmail.com> <710764ea0812281200u4182658x1099339d1f3eeed0@mail.gmail.com> Message-ID: <710764ea0812281238y430c7217u4d4d2441b8ebcd83@mail.gmail.com> ---------- Forwarded message ---------- From: Pow Joshi Date: 2008/12/28 Subject: Re: Gewtting the concentration of PCR product and cloning To: aawara@pontiff-playground.org >> > Dear all! >> > Please read carefully what DK wrote before pointing fingers at me! >> > Mr. Aawara is also repeating the same thing.[PCR products amplified >> > with a proof-reading polymerase must be A-tailed ]. Please note that, if >> the enzyme has proof reading activity, it will remove 3'A and TA cloning >> cannot be done. And, it is Taq's property to put an extra A at 3' which has >> nothing to do with its proof reading. >> >> The problem is with your comprehension of English. Let me clarify: >> >> If one wrote, "PCR products .... ARE A-tailed", then it would indicate >> that products made with a proof-reading polymerase are already A-tailed. >> >> On the other hand, if one writes "PCR products .... MUST BE A-tailed", >> then >> it indicates that the products made with a proof-reading polymerase are >> NOT A-tailed, and therefore MUST BE A-tailed for them to be cloned into a >> TA-vector. >> >> Hope that helps you ..... > > > I agree with AC .... which is why you need to re-read Dima's reply. You can > 3' A-tail post-PCR, if that's what you wish to know., by just incubating > with regular Taq at 72degC... You would find that in the TOPO manual if you > read it carefully. It is exactly what DIma said when he wrote " if you used > proofreading polymerase, it would have to be A-tailed first. If Taq and the > like, no need to."!!! ..... > > Sure this is an open forum, and defintely not a place for bashing up, which > I would say, you were an instigator, a provocateur, with your absolutely > wrong statement of "What DK says is incorrect!" before you even bothered to > comprehend what was written (by Dima). We do assume in this forum that > people understand english as a language. Clearly, though you don't! > > Check your premises before you make any statements, is the first rule of > Logic. You want to read Aristotle perhaps ;) > >> >> >> AC >> -- >> Email: echo 36434455860060025978157675027927670979097959886449930P | dc >> _______________________________________________ >> Methods mailing list >> Methods@net.bio.net >> http://www.bio.net/biomail/listinfo/methods >> > > From aawara from pontiff-playground.org Sun Dec 28 17:23:40 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Sun Dec 28 18:54:44 2008 Subject: what is the strongest protein-protein interaction? References: <3kIUk.8581$ev2.3155@newsfe12.iad> Message-ID: In <3kIUk.8581$ev2.3155@newsfe12.iad>, DK wrote: > To the OP, I would suggest artificial coiled coils. Basically, a pair of > 21 aa peptides used as interacting tags. One is very, very basic, > another is very, very acidic - both conforming to the canonical coiled > coil heptade repeat, so the only coiled coil formed is a heterodimer. > I don't have a ref. but have a xerox somewhere at work. The affinity > of such pair is subnanomolar. I've used a modified version of the GCN4 leucine zipper for this purpose. Sadly, I don't have the reference handy - I am at home, but this worked very well to induce dimerization. If it helps any, the paper that I got the sequence from was from Peter Kim's lab, and published in Biochemistry. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From davidminde from gmail.com Sun Dec 28 19:49:06 2008 From: davidminde from gmail.com (David-Paul Minde) Date: Mon Dec 29 13:19:49 2008 Subject: Gewtting the concentration of PCR product and cloning In-Reply-To: <710764ea0812281238y430c7217u4d4d2441b8ebcd83@mail.gmail.com> References: <710764ea0812281155u17dcd31l1364196ab1a941fe@mail.gmail.com> <710764ea0812281200u4182658x1099339d1f3eeed0@mail.gmail.com> <710764ea0812281238y430c7217u4d4d2441b8ebcd83@mail.gmail.com> Message-ID: <123243900812281649j4a1eb9d9pa1e0b1b0df01a780@mail.gmail.com> > > > > Check your premises before you make any statements, is the first rule of > > Logic. You want to read Aristotle perhaps ;) > I recommend the (classical) greek original. That takes a bit longer and > might refresh some minds more profoundly :) > >> > >> AC > >> -- > >> Email: echo 36434455860060025978157675027927670979097959886449930P | dc > >> _______________________________________________ > >> 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 > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 I never think of the future. It comes soon enough. Albert Einstein Res severa verum gaudium. (~true delight is a severe issue) Seneca From estebangrasso from gmail.com Mon Dec 29 14:26:33 2008 From: estebangrasso from gmail.com (egrasso) Date: Mon Dec 29 17:09:47 2008 Subject: need plasmid map or sequence - PT81-Luc Message-ID: Hi, I need the plasmid map or sequence (i prefer the sequence) of the plasmid pT81-Luc. It is a Luciferase reporter plasmid with a minimal thymidine kinase promoter. I already tried ATCC, entrez, google and I didn't find neither the map nor the sequence. All I found was a reference to a paper from 1988. Thanks Esteban From aawara from pontiff-playground.org Tue Dec 30 10:24:00 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Tue Dec 30 13:05:02 2008 Subject: need plasmid map or sequence - PT81-Luc References: Message-ID: In , DK wrote: > In article , egrasso wrote: >>Hi, I need the plasmid map or sequence (i prefer the sequence) of the >>plasmid pT81-Luc. It is a Luciferase reporter plasmid with a minimal >>thymidine kinase promoter. I already tried ATCC, entrez, google and I >>didn't find neither the map nor the sequence. All I found was a >>reference to a paper from 1988. > > ATCC sells the vector (cat. # 37584) - maybe write them asking for > a deposited sequence. Alternatively, ask the depositor: > Steven Nordeen, Steve.Nordeen@UCDenver.edu - maybe he still > has the sequence somewhere on floppy disk :-) My recollection is that Nordeen constructed his plasmids as derivatives of pSVL (de Wet et al., MCB 7:725, 1987). The details of construction are described in Nordeen's Biotechniques paper (6:454, 1988). I have reconstructed a sequence for pSVL - which I would be happy to email you. Using it, and Nordeen's construction details, it should be relatively simple to reconstruct the sequence of pT81luc. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc