From campanellj from mail.montclair.edu Tue Jul 1 18:34:50 2008 From: campanellj from mail.montclair.edu (James J. Campanella) Date: Wed Jul 2 12:55:45 2008 Subject: pCambia1201 question Message-ID: Dear fellow workers in the mines of science, I have been tryingto work with the pCambia1201 plasmid for the lastseveral weeks, but ithas been driving my lab a bit crazy. My problemis that it does not wantto digest with most endonucleases we've tried.I simply can not use theplasmid if I can't cut it, so I have aproblem. It was suggested to meto try transferring the plasmid to DH5cells, which I did to no avail--the resultant plasmid still does notcut with almost every enzyme in mylibrary. I was finally able to getit to cut with XhoI and PvuII, buteven those were only partialdigests. Does anybody have anyadvice on working with this thing? The literaturedoes not suggest thatthere is even a problem, and the group thatengineered it could onlysuggest "trying all your enzymes until youfind one that cuts", which isa bit impractical. Thanks for any help, Jim Campanella ------------------- James J. Campanella, Associate Professor, Department of Biology and Molecular Biology Montclair State University 1 Normal Avenue Montclair, NJ 07043 Alternate email address: jcamp@alumni.uchicago.edu Ph: 973-655-4097 Fax: 973-655-7047 From engelbert_buxbaum from hotmail.com Thu Jul 3 09:14:53 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Thu Jul 3 11:57:50 2008 Subject: protocol for membrane receptor isolation References: Message-ID: Am 25.06.2008, 06:18 Uhr, schrieb WS : > Just wonder if membrane proteins get solubilized without detegent? No. Membrane attached proteins can be washed of membranes by high salt (100 mM Na2CO3 pH 11.5 or 250 mM KI or 2 M NaBr), but transmembrane proteins require detergents. > Will eg 0.5% TX100 disrupt the antibody-recptor complex? That depends on the nature and concentration of the detergent and on the antibody-antigen interactions. Note however that in immunoprecipitation studies even relatively harsh detergents like SDS are frequently used to ensure specificity. For example, in their booklet on Pansorbin Calbiochem recommends the following "superwash buffer": 100 mM NaCl, 50 mM Tris-HCl pH 7.4, 2 mM EDTA, 2% Triton X-100, 0.5% SDS. Of course, not all antibodies can be used with these conditions, but some titration should give you detergent concentrations where specific binding survives with minimal background. From josenet from tiscali.co.uk Thu Jul 3 17:23:02 2008 From: josenet from tiscali.co.uk (Jose de las Heras) Date: Fri Jul 4 10:43:15 2008 Subject: southern size standards References: Message-ID: <6d51q9Ftk7oU1@mid.individual.net> "Ed Siefker" wrote in message news:mailman.560.1214432240.3533.methods@net.bio.net... > I'm looking for a DNA size standard I can use with southern blots. I > don't > want to deal with radioactive gel apparatus, so I don't want to end label > my ladder before I run the gel. The ideal solution would be something > dyed > so I could see with the naked eye on the membrane, and mark the sizes with > radioactive ink. Is there something like this available? > > Here's an idea, I can see the DNA ladder in my gel under UV. Could I > inject > a small amount of P32 into each size standard, and then transfer so the > size > standards would be visible on the membrane? > > I've seen protocols for making size standards from lambda DNA, but these > require probing with lambda DNA. If I do this, would I include the lambda > probe in the hybridization with my probe? Do I need to reprobe with the > lambda probe? I tend to simply add a *minute* amount of the ladder to my probe during the labelling reaction. Then you see the ladder as well as your probe... but of course, you have to be sure there won't be crosshybridisation. Jose From k.schink_nospam_ from _nospam_gmail.com Fri Jul 4 04:51:23 2008 From: k.schink_nospam_ from _nospam_gmail.com (Kay Schink) Date: Fri Jul 4 10:43:49 2008 Subject: southern size standards In-Reply-To: References: Message-ID: Ed Siefker schrieb: > I'm looking for a DNA size standard I can use with southern blots. I don't > want to deal with radioactive gel apparatus, so I don't want to end label > my ladder before I run the gel. The ideal solution would be something dyed > so I could see with the naked eye on the membrane, and mark the sizes with > radioactive ink. Is there something like this available? > > Here's an idea, I can see the DNA ladder in my gel under UV. Could I > inject > a small amount of P32 into each size standard, and then transfer so the > size > standards would be visible on the membrane? > > I've seen protocols for making size standards from lambda DNA, but these > require probing with lambda DNA. If I do this, would I include the lambda > probe in the hybridization with my probe? Do I need to reprobe with the > lambda probe? > In our lab, we have frequently observed cross-hybridisation of labelled probes with the DNA Ruler Ladder Mix from Fermentas (no affiliation). Interestingly, this happens only with this marker, but not with others like Lamba-Pst - so you could give it a try if this wouks with your probes, too. We also usually photograph our gels with a ruler next to it, so by measuring the distance from the wels to our band, we can interpolate the sizes. Good luck Kay From virashkgupta from gmail.com Sat Jul 5 07:20:54 2008 From: virashkgupta from gmail.com (Virash Gupta) Date: Sat Jul 5 11:27:38 2008 Subject: Methods Digest, Vol 38, Issue 2-pCambia 1201 Message-ID: It is really depressing when you donot get the desired results. If the plasmid is good sometime you loose sites that are involved in restriction followed by ligation, others remain unaffected. It also happens if the plasmid is isolated using phenol chloroform extraction using old preparation of phenol ( gets oxidized with time and makes restriction sites resistant to cut). I suggest go back to your original plasmid which you acquired from any other source, transform into DH5-alpha and isolate plasmid using alkaline lysis method ( solution P2 should not be old- absorbs CO2 from atmosphere to result decrease in pH). This will also enhance plasmid yield. (Do not use plasmid purification kit based upon glass memebrane or glass powder.) Try cutting this plasmid.I am sure if recieved right plasmid, you will be successful in restricting all sites. It must work now and if does not just forget and order for new preparation from CAMBIA- (available freely as DNA spot on filter paper- Cut the disk, elute DNa with TE use eluent for transforming DH5-Alpha). Tell me about results. Alternately move to pCambia 130. All the best. On 7/3/08, methods-request@oat.bio.indiana.edu wrote: > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. pCambia1201 question (James J. Campanella) > 2. Re: protocol for membrane receptor isolation > (Dr Engelbert Buxbaum) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Tue, 01 Jul 2008 19:34:50 -0400 > From: "James J. Campanella" > Subject: pCambia1201 question > To: methods@magpie.bio.indiana.edu > Message-ID: > Content-Type: text/plain; charset=us-ascii > > Dear fellow workers in the mines of science, > > I have been tryingto work with the pCambia1201 plasmid for the lastseveral weeks, but ithas been driving my lab a bit crazy. My problemis that it does not wantto digest with most endonucleases we've tried.I simply can not use theplasmid if I can't cut it, so I have aproblem. It was suggested to meto try transferring the plasmid to DH5cells, which I did to no avail--the resultant plasmid still does notcut with almost every enzyme in mylibrary. I was finally able to getit to cut with XhoI and PvuII, buteven those were only partialdigests. > > Does anybody have anyadvice on working with this thing? The literaturedoes not suggest thatthere is even a problem, and the group thatengineered it could onlysuggest "trying all your enzymes until youfind one that cuts", which isa bit impractical. > > Thanks for any help, > > Jim Campanella > > ------------------- > > James J. Campanella, > Associate Professor, > Department of Biology and Molecular Biology > Montclair State University > 1 Normal Avenue > Montclair, NJ 07043 > > Alternate email address: jcamp@alumni.uchicago.edu > > Ph: 973-655-4097 > Fax: 973-655-7047 > > > > ------------------------------ > > Message: 2 > Date: Thu, 03 Jul 2008 10:14:53 -0400 > From: "Dr Engelbert Buxbaum" > Subject: Re: protocol for membrane receptor isolation > To: methods@net.bio.net > Message-ID: > Content-Type: text/plain; format=flowed; delsp=yes; > charset=iso-8859-15 > > Am 25.06.2008, 06:18 Uhr, schrieb WS : > > > Just wonder if membrane proteins get solubilized without detegent? > > No. Membrane attached proteins can be washed of membranes by high salt > (100 mM Na2CO3 pH 11.5 or 250 mM KI or 2 M NaBr), but transmembrane > proteins require detergents. > > > Will eg 0.5% TX100 disrupt the antibody-recptor complex? > > That depends on the nature and concentration of the detergent and on the > antibody-antigen interactions. Note however that in immunoprecipitation > studies even relatively harsh detergents like SDS are frequently used to > ensure specificity. For example, in their booklet on Pansorbin Calbiochem > recommends the following "superwash buffer": 100 mM NaCl, 50 mM Tris-HCl > pH 7.4, 2 mM EDTA, 2% Triton X-100, 0.5% SDS. Of course, not all > antibodies can be used with these conditions, but some titration should > give you detergent concentrations where specific binding survives with > minimal background. > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 38, Issue 2 > ************************************** > -- 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 Sat Jul 5 07:23:56 2008 From: virashkgupta from gmail.com (Virash Gupta) Date: Sat Jul 5 11:27:44 2008 Subject: Methods Digest, Vol 38, Issue 2 In-Reply-To: <200807031705.m63H5QO26042@net.bio.net> References: <200807031705.m63H5QO26042@net.bio.net> Message-ID: One thing more- retransform. Pick up 5-6 single clones (Blue clnes on X-Gal/IPTG) culture individually and isolate plasmid from each culture. Compare restriction and select the best one. On 7/3/08, methods-request@oat.bio.indiana.edu wrote: > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. pCambia1201 question (James J. Campanella) > 2. Re: protocol for membrane receptor isolation > (Dr Engelbert Buxbaum) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Tue, 01 Jul 2008 19:34:50 -0400 > From: "James J. Campanella" > Subject: pCambia1201 question > To: methods@magpie.bio.indiana.edu > Message-ID: > Content-Type: text/plain; charset=us-ascii > > Dear fellow workers in the mines of science, > > I have been tryingto work with the pCambia1201 plasmid for the lastseveral weeks, but ithas been driving my lab a bit crazy. My problemis that it does not wantto digest with most endonucleases we've tried.I simply can not use theplasmid if I can't cut it, so I have aproblem. It was suggested to meto try transferring the plasmid to DH5cells, which I did to no avail--the resultant plasmid still does notcut with almost every enzyme in mylibrary. I was finally able to getit to cut with XhoI and PvuII, buteven those were only partialdigests. > > Does anybody have anyadvice on working with this thing? The literaturedoes not suggest thatthere is even a problem, and the group thatengineered it could onlysuggest "trying all your enzymes until youfind one that cuts", which isa bit impractical. > > Thanks for any help, > > Jim Campanella > > ------------------- > > James J. Campanella, > Associate Professor, > Department of Biology and Molecular Biology > Montclair State University > 1 Normal Avenue > Montclair, NJ 07043 > > Alternate email address: jcamp@alumni.uchicago.edu > > Ph: 973-655-4097 > Fax: 973-655-7047 > > > > ------------------------------ > > Message: 2 > Date: Thu, 03 Jul 2008 10:14:53 -0400 > From: "Dr Engelbert Buxbaum" > Subject: Re: protocol for membrane receptor isolation > To: methods@net.bio.net > Message-ID: > Content-Type: text/plain; format=flowed; delsp=yes; > charset=iso-8859-15 > > Am 25.06.2008, 06:18 Uhr, schrieb WS : > > > Just wonder if membrane proteins get solubilized without detegent? > > No. Membrane attached proteins can be washed of membranes by high salt > (100 mM Na2CO3 pH 11.5 or 250 mM KI or 2 M NaBr), but transmembrane > proteins require detergents. > > > Will eg 0.5% TX100 disrupt the antibody-recptor complex? > > That depends on the nature and concentration of the detergent and on the > antibody-antigen interactions. Note however that in immunoprecipitation > studies even relatively harsh detergents like SDS are frequently used to > ensure specificity. For example, in their booklet on Pansorbin Calbiochem > recommends the following "superwash buffer": 100 mM NaCl, 50 mM Tris-HCl > pH 7.4, 2 mM EDTA, 2% Triton X-100, 0.5% SDS. Of course, not all > antibodies can be used with these conditions, but some titration should > give you detergent concentrations where specific binding survives with > minimal background. > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 38, Issue 2 > ************************************** > -- 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 utsuxs from hotmail.com Tue Jul 8 20:02:11 2008 From: utsuxs from hotmail.com (utsuxs@hotmail.com) Date: Tue Jul 8 21:02:02 2008 Subject: RNA on denaturing gels References: Message-ID: On Jun 27, 5:35 am, "Simone Marker" wrote: > Hi, > > since I want to do a northern blot, I have to perform a denaturing agaose > gel electrophorese with total RNA of Paramecium. > > My problem is, that I don't even get the typical bands (28S and 18S rRNA) > for total eukaryotic RNA. I isolated the RNA with Trizol and prepared a 1,1% > agarose gel with Formaldehyd (3-4ml 37% per 40ml gel solution) in a northern > running buffer (20mM MOPS, 5 mM NaAc, 1mM EDTA). The run was at 50-70V. > I denatured the RNA samples at 65?C for 5-10 min. > > I used a ssRNA ladder in its original purchased loading buffer (7M urea, > ficoll,...), which showed a perfect run in this gel. In contrast, my RNA > samples in this loading buffer showed a pattern of different bands with one > very dominant (at ca 3500 nt). > When I used another common loading buffer receipe(50% formamide, 25% > formaldehyde,...), I got a smear and some weak bands. I can nearly exclude > that the RNA that I used was not severely degraded, since I had it on a > bioanalyser a few month ago (stored at -70?C). > > What might be wrong in my system? > Thank you very much! > simone These are just random thoughts. You sure you have RNA and not chewed up DNA? Just to confirm, you got two bands/peaks on the bioanalyser? I forgot where tRNA ends up but that could be the 3500nt band. And contamination. Your ingredients and equipment, including your gel box might contaminated with RNase which is hell to get rid of. Wouldn't matter if your RNA is RNA and intact, you drop it in an RNase contaminated environment, well so long RNA. From eudolin from googlemail.com Tue Jul 8 22:24:42 2008 From: eudolin from googlemail.com (Lara) Date: Wed Jul 9 10:05:38 2008 Subject: Trypsin/EDTA Message-ID: Hello there folks, I use for some endothelial cells trpsin/edta and according to the protocol I have to pipette in 5 ml trypsin/edta and then immediately remove 4.5 ml and leave the rest for a couple of minutes. Does anyone know why? Isn't this simply a waste of trypsin? Furthermore, is there any place where I can find detailed info about trypsin in particular? Thanks for any info. Cheers, Lara From nobody from nospam.not Wed Jul 9 05:39:33 2008 From: nobody from nospam.not (Han) Date: Wed Jul 9 10:05:51 2008 Subject: Trypsin/EDTA References: Message-ID: dk@no.email.thankstospam.net (DK) wrote in news:BcXck.350$BC4.114@newsfe07.lga: > In article > , > Lara wrote: >>Hello there folks, >> >>I use for some endothelial cells trpsin/edta and according to the >>protocol I have to pipette in 5 ml trypsin/edta and then immediately >>remove 4.5 ml > > Not immediately. Slosh the dish few times and *then* immediately > remove. > >>and leave the rest for a couple of minutes. Does anyone >>know why? Isn't this simply a waste of trypsin? > > No, it's not. There is always some medium left - almost 0.5 ml > from 10 cm dish, typically. So it makes a huge difference > whether you dilute it with 0.5 ml or 5 ml of trypsin/EDTA. > Remember that most media contain serum and serum contains > powerful trypsin inhibitor - you want to add enough trypsin to > titrate it out. Likewise, medium contains Mg and Ca - you want > to add enough EDTA to chelate them or cells won't detach. > You could just wash with EDTA and add little trypsin but the > cost of doing in sterile pipettes and time exceeds the cost of > the crude trypsin preparation used in cell culture. > >>Furthermore, is there any place where I can find detailed info about >>trypsin in particular? Thanks for any info. > > Any of the hundreds of the cell culture practical guide books. > Some cell lined don't even require trypsin - just EDTA is > enough. > > DK > I agree with DK. On the other hand, we generally use collagenase, a rather drude preparation with trypsin as a major contaminant. Because EC are generall grown on a gelatin coating, you'll need a bit more enzyme activity than when detaching cell lines from just plastic. It may even depend on how you lay down the gelatin, coat 1 hr at RT, vs overnight at 4C. -- Best regards Han email address is invalid From pjie2 from cam.ac.uk Wed Jul 9 05:54:04 2008 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Wed Jul 9 10:05:57 2008 Subject: RNA on denaturing gels In-Reply-To: References: Message-ID: On Tue, 8 Jul 2008, utsuxs@hotmail.com wrote: > > These are just random thoughts. > I forgot where tRNA ends up but that could be the 3500nt band. Transfer RNAs are ~75 bp, so.... no. > And contamination. Your ingredients and equipment, including your gel > box might contaminated with RNase which is hell to get rid of. > Wouldn't matter if your RNA is RNA and intact, you drop it in an RNase > contaminated environment, well so long RNA. Yes, but the OP says his ssRNA ladder ran fine, which argues against degradation in the gel. To the OP - are you sure what size ribosomal bands you're expecting from Paramecium? I strongly doubt it'll be the exact same pattern/size you'd expect from mammalian cells. Peter From eckofoid from ucdavis.edu Wed Jul 9 15:31:13 2008 From: eckofoid from ucdavis.edu (Eric Kofoid) Date: Wed Jul 9 17:06:29 2008 Subject: PCRBoost Message-ID: I just used my free sample of "PCRBoost" from Biomatrica. To my surprise, it worked and I got the promised >5X increase in product level. The problem is, the stuff is expensive and I would be unable to justify it for anything other that very recalcitrant reactions. Does anybody know what's in it? Cheers, Eric. -- Eric Kofoid Microbiology/CBS, UC Davis -------------------------- "Measure what is measurable, and make measurable what is not so." - Galileo Galilei From novalidaddress from nurfuerspam.de Wed Jul 9 18:29:10 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Jul 9 22:25:59 2008 Subject: PCRBoost References: Message-ID: Trehalose? Ectoine? Hydroxyectoine? maybe have a look at http://www.bitop.de - unfortunately, only in German. Wo From novalidaddress from nurfuerspam.de Wed Jul 9 18:30:38 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Jul 9 22:26:03 2008 Subject: PCRBoost References: Message-ID: Hi Dima, you get 110% > P.S. If you decide to try trehalose, buy it as pure as possible > and make it "molecular biology grade" by heating at 100C > for 20 min. Best regards, Wo From eudolin from googlemail.com Wed Jul 9 22:36:53 2008 From: eudolin from googlemail.com (Lara) Date: Thu Jul 10 11:50:46 2008 Subject: Trypsin/EDTA References: Message-ID: <3fad1675-2197-41bb-a5d0-638a4562f9c5@a1g2000hsb.googlegroups.com> Dear DK and Han, thanks so much for your helpful feedback. Indeed, now I understand it is not a waste of either Trypsin or EDTA. With responses like this newsgroups get another dimension in science. Thank a lot again! Yours, Lara On Jul 8, 5:24?pm, Lara wrote: > Hello there folks, > > I use for some endothelial cells trpsin/edta and according to the > protocol I have to pipette in 5 ml trypsin/edta and then immediately > remove 4.5 ml and leave the rest for a couple of minutes. Does anyone > know why? Isn't this simply a waste of trypsin? > > Furthermore, is there any place where I can find detailed info about > trypsin in particular? Thanks for any info. > > Cheers, > Lara From ahmadalbasheer from hotmail.com Wed Jul 9 23:36:19 2008 From: ahmadalbasheer from hotmail.com (Ahmad Al-Basheer) Date: Thu Jul 10 11:50:52 2008 Subject: Karyotyping protocol In-Reply-To: References: Message-ID: hello, can any body help me how can I get the karyotyping protocol or how to make it in my lab and what I need, please. Thank you all _________________________________________________________________ Need to know now? Get instant answers with Windows Live Messenger. http://www.windowslive.com/messenger/connect_your_way.html?ocid=TXT_TAGLM_WL_messenger_072008 From legatek from hotmail.com Thu Jul 10 00:09:14 2008 From: legatek from hotmail.com (Kyle Legate) Date: Thu Jul 10 11:51:01 2008 Subject: Trypsin/EDTA In-Reply-To: <3fad1675-2197-41bb-a5d0-638a4562f9c5@a1g2000hsb.googlegroups.com> References: <3fad1675-2197-41bb-a5d0-638a4562f9c5@a1g2000hsb.googlegroups.com> Message-ID: <6dljquF36jrlU1@mid.individual.net> Lara wrote: > Dear DK and Han, > > thanks so much for your helpful feedback. Indeed, now I understand it > is not a waste of either Trypsin or EDTA. With responses like this > newsgroups get another dimension in science. Thank a lot again! > It most certainly is a waste of trypsin/EDTA. If what DK says is true, and the extra solution is to dilute serum-containing medium, you can achieve the same goal by washing the plate once with PBS and then adding a smaller volume of trypsin. Here is what I do for 10 cm plates or 75 cm2 flasks: aspirate medium, wash once with 10 ml PBS, aspirate, add 1 ml trypsin/EDTA and place in the incubator for 5 min, or until cells detach. I have never had a problem. From jleonard.iii from gmail.com Thu Jul 10 12:43:03 2008 From: jleonard.iii from gmail.com (John Leonard) Date: Thu Jul 10 14:06:38 2008 Subject: IP Problem Message-ID: <83d01d5d0807101043k23800833pab2eb77f70eae006@mail.gmail.com> Hello all, I'm looking to perform an IP Western of a protein that runs at around 50-55kD on an SDS-PAGE gel - approximately the same as my antibody's heavy chain. As such, it could be very difficult to detect presence of my protein by IP Western, since the antibody is generally eluted along with the antigen. I have read protocols which suggest to cross-link your antibody to the protein A/G beads prior to IP, however I'm using rabbit antiserum (not immunoaffinity purified), so I don't know whether this will be effective. A co-worker of mine suggested running a non-denaturing gel instead to observe either a) separation of the protein of interest and the antibody by nature of different charge/size ratios; or b) differential shifting of the antibody band (when compared with a negative/positive controls) to indicate interaction with my protein of interest. I don't know how effective this would be, but in that case I would need a good NON-DENATURING elution buffer to detach my antigen from the bead complex. The whole purpose of this experiment is to determine whether my antiserum will be sufficient for IPs, ChIP, etc or whether I should immunoaffinity purify it. (I'll be pre-clearing with normal rabbit serum to reduce non-specific binding). If anyone has comments/suggestions about this, or knows of A GOOD NON-DENATURING ELUTION BUFFER, I'd love to hear it! Thanks, John From arnec from bio.umass.edu Thu Jul 10 15:02:35 2008 From: arnec from bio.umass.edu (Arne Christensen) Date: Thu Jul 10 19:53:16 2008 Subject: IP Problem In-Reply-To: <83d01d5d0807101043k23800833pab2eb77f70eae006@mail.gmail.com> References: <83d01d5d0807101043k23800833pab2eb77f70eae006@mail.gmail.com> Message-ID: <48766ADB.8080305@bio.umass.edu> Are there any antibodies raised against your protein in species other than rabbit (e.g. monoclonal, guinea pig)? Using an antibody from a different species to detect would be the simplest solution. John Leonard wrote: > Hello all, > > I'm looking to perform an IP Western of a protein that runs at around > 50-55kD on an SDS-PAGE gel - approximately the same as my antibody's heavy > chain. As such, it could be very difficult to detect presence of my protein > by IP Western, since the antibody is generally eluted along with the > antigen. I have read protocols which suggest to cross-link your antibody to > the protein A/G beads prior to IP, however I'm using rabbit antiserum (not > immunoaffinity purified), so I don't know whether this will be effective. > A co-worker of mine suggested running a non-denaturing gel instead to > observe either a) separation of the protein of interest and the antibody by > nature of different charge/size ratios; or b) differential shifting of > the antibody band (when compared with a negative/positive controls) to > indicate interaction with my protein of interest. I don't know how > effective this would be, but in that case I would need a good NON-DENATURING > elution buffer to detach my antigen from the bead complex. > > The whole purpose of this experiment is to determine whether my antiserum > will be sufficient for IPs, ChIP, etc or whether I should immunoaffinity > purify it. (I'll be pre-clearing with normal rabbit serum to reduce > non-specific binding). > > If anyone has comments/suggestions about this, or knows of A GOOD > NON-DENATURING ELUTION BUFFER, I'd love to hear it! > > > Thanks, > > John > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > -- __ Arne K Christensen Postdoctoral Research Associate University of Massachusetts, Amherst USGS Conte Anadromous Fish Research Center One Migratory Way, PO Box 796 Turners Falls, MA 01376 Email: arnec@bio.umass.edu Phone: (413) 863-3827 Fax: (413) 863-9810 URL: www.biobog.com From allison from nospam.com Thu Jul 10 15:33:39 2008 From: allison from nospam.com (allisonh) Date: Thu Jul 10 19:53:22 2008 Subject: IP Problem In-Reply-To: References: Message-ID: <48766b54$0$16247$9a6e19ea@news.newshosting.com> John Leonard wrote: > Hello all, > > I'm looking to perform an IP Western of a protein that runs at around > 50-55kD on an SDS-PAGE gel - approximately the same as my antibody's heavy > chain. As such, it could be very difficult to detect presence of my protein > by IP Western, since the antibody is generally eluted along with the > antigen. I have read protocols which suggest to cross-link your antibody to > the protein A/G beads prior to IP, however I'm using rabbit antiserum (not > immunoaffinity purified), so I don't know whether this will be effective. > A co-worker of mine suggested running a non-denaturing gel instead to > observe either a) separation of the protein of interest and the antibody by > nature of different charge/size ratios; or b) differential shifting of > the antibody band (when compared with a negative/positive controls) to > indicate interaction with my protein of interest. I don't know how > effective this would be, but in that case I would need a good NON-DENATURING > elution buffer to detach my antigen from the bead complex. > > The whole purpose of this experiment is to determine whether my antiserum > will be sufficient for IPs, ChIP, etc or whether I should immunoaffinity > purify it. (I'll be pre-clearing with normal rabbit serum to reduce > non-specific binding). > > If anyone has comments/suggestions about this, or knows of A GOOD > NON-DENATURING ELUTION BUFFER, I'd love to hear it! > > > Thanks, > > John If you run a regular SDS gel without reducing agent in the sample buffer (ie, no beta-mercaptoethanol) then the antibodies will run at 150K. If your protein is a single unit then it will still run at about 50-55K. Allison From akhan357 from sbcglobal.net Thu Jul 10 17:42:06 2008 From: akhan357 from sbcglobal.net (AK) Date: Thu Jul 10 19:53:26 2008 Subject: IP Problem References: Message-ID: I had same problem once added with my co-IP protein died upon heating the sample. 6M urea in sample buffer did the trick without heating which sent the IgG bands close to 100kDa. if I remember correctly I also added 10mM DTT that apparently did not effect shift of IgG to higher mol wt signal. good luck. AK "John Leonard" wrote in message news:mailman.701.1215716798.3533.methods@net.bio.net... > Hello all, > > I'm looking to perform an IP Western of a protein that runs at around > 50-55kD on an SDS-PAGE gel - approximately the same as my antibody's heavy > chain. As such, it could be very difficult to detect presence of my > protein > by IP Western, since the antibody is generally eluted along with the > antigen. I have read protocols which suggest to cross-link your antibody > to > the protein A/G beads prior to IP, however I'm using rabbit antiserum (not > immunoaffinity purified), so I don't know whether this will be effective. > A co-worker of mine suggested running a non-denaturing gel instead to > observe either a) separation of the protein of interest and the antibody > by > nature of different charge/size ratios; or b) differential shifting of > the antibody band (when compared with a negative/positive controls) to > indicate interaction with my protein of interest. I don't know how > effective this would be, but in that case I would need a good > NON-DENATURING > elution buffer to detach my antigen from the bead complex. > > The whole purpose of this experiment is to determine whether my antiserum > will be sufficient for IPs, ChIP, etc or whether I should immunoaffinity > purify it. (I'll be pre-clearing with normal rabbit serum to reduce > non-specific binding). > > If anyone has comments/suggestions about this, or knows of A GOOD > NON-DENATURING ELUTION BUFFER, I'd love to hear it! > > > Thanks, > > John From eudolin from googlemail.com Fri Jul 11 02:40:43 2008 From: eudolin from googlemail.com (Lara) Date: Fri Jul 11 13:31:17 2008 Subject: Trypsin/EDTA References: <3fad1675-2197-41bb-a5d0-638a4562f9c5@a1g2000hsb.googlegroups.com> <6dljquF36jrlU1@mid.individual.net> <4Ahdk.2981$dc7.927@newsfe06.lga> <6dod90F3gmilU1@mid.individual.net> Message-ID: <747775a1-7d11-493c-86c3-5d6c547c68ab@l42g2000hsc.googlegroups.com> > Washing with PBS is preferable because it more effectively removes the > inhibitors (as you don't leave any behind in the flask when you aspirate > all the PBS), and washing with PBS first removes dead cells and debris > much more effectively. I am not denying any of what has been written so far but you have to apologize if I might ask you what constitutes chemically the fact that it is more "effective". The expression more effective seems to me a bit too broad or if you want too vague. After trypsinization you neutralize with HBBS and remove so therefore you leave no traces behind. The question here is not what is more economical but what does better to the cells and since we all are interested in what is best for our cells we stand on the same site, right? Lara From josenet from tiscali.co.uk Thu Jul 10 19:56:51 2008 From: josenet from tiscali.co.uk (Jose de las Heras) Date: Fri Jul 11 13:31:37 2008 Subject: Trypsin/EDTA References: <3fad1675-2197-41bb-a5d0-638a4562f9c5@a1g2000hsb.googlegroups.com> <6dljquF36jrlU1@mid.individual.net> <4Ahdk.2981$dc7.927@newsfe06.lga> Message-ID: <6dnpenF3hv2aU1@mid.individual.net> "DK" wrote in message news:4Ahdk.2981$dc7.927@newsfe06.lga... > > "You could just wash with EDTA and add little trypsin but the > cost of doing it in sterile pipettes and time exceeds the cost of > the crude trypsin preparation used in cell culture." > > DK I normally just wash with a bit of PBS and then put a small amount of trypsin, like Kyle said, but that's because I usually deal with only a few flasks at any one time. When you are facing several stacks containing a hundred or so dishes, adding extra trypsin to dilute remaining medium and removing most of it is a much more palatable approach... Jose From legatek from hotmail.com Fri Jul 11 01:30:45 2008 From: legatek from hotmail.com (Kyle Legate) Date: Fri Jul 11 13:31:42 2008 Subject: Trypsin/EDTA In-Reply-To: <4Ahdk.2981$dc7.927@newsfe06.lga> References: <3fad1675-2197-41bb-a5d0-638a4562f9c5@a1g2000hsb.googlegroups.com> <6dljquF36jrlU1@mid.individual.net> <4Ahdk.2981$dc7.927@newsfe06.lga> Message-ID: <6docvlF3j9kjU1@mid.individual.net> DK wrote: > In article <6dljquF36jrlU1@mid.individual.net>, Kyle Legate wrote: >> Lara wrote: >>> Dear DK and Han, >>> >>> thanks so much for your helpful feedback. Indeed, now I understand it >>> is not a waste of either Trypsin or EDTA. With responses like this >>> newsgroups get another dimension in science. Thank a lot again! >>> >> It most certainly is a waste of trypsin/EDTA. If what DK says is true > > Which is certainly true: it is an undeniable fact that serum contains > alpha2-macrglobulin and medium contains ~ 2 mM of Mg2+ and > Ca2+. > I'm not denying it. From legatek from hotmail.com Fri Jul 11 01:35:44 2008 From: legatek from hotmail.com (Kyle Legate) Date: Fri Jul 11 13:31:46 2008 Subject: Trypsin/EDTA In-Reply-To: <4Ahdk.2981$dc7.927@newsfe06.lga> References: <3fad1675-2197-41bb-a5d0-638a4562f9c5@a1g2000hsb.googlegroups.com> <6dljquF36jrlU1@mid.individual.net> <4Ahdk.2981$dc7.927@newsfe06.lga> Message-ID: <6dod90F3gmilU1@mid.individual.net> DK wrote: > In article <6dljquF36jrlU1@mid.individual.net>, Kyle Legate wrote: >> Lara wrote: >>> Dear DK and Han, >>> >>> thanks so much for your helpful feedback. Indeed, now I understand it >>> is not a waste of either Trypsin or EDTA. With responses like this >>> newsgroups get another dimension in science. Thank a lot again! >>> >> It most certainly is a waste of trypsin/EDTA. If what DK says is true > > Which is certainly true: it is an undeniable fact that serum contains > alpha2-macrglobulin and medium contains ~ 2 mM of Mg2+ and > Ca2+. > I'm not denying it. Clearly adding extra trypsin and then removing it dilutes inhibiting factors, but this practice was probably first suggested by a company to sell more product. It is not the better way. Washing with PBS is preferable because it more effectively removes the inhibitors (as you don't leave any behind in the flask when you aspirate all the PBS), and washing with PBS first removes dead cells and debris much more effectively. From SBrown from ccia.unsw.edu.au Fri Jul 11 04:22:02 2008 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Fri Jul 11 13:31:51 2008 Subject: Cre recombinase question Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A02972A0A@mail01.ccia.local> Hi, I have performed a few digests on what I am pretty certain is a Cre plasmid however none of the fragments match up to the plasmid map. To validate that i do infact have Cre, I was thinking of doing a cotransfection, transfecting one population of 293's with pCre and another with a trap construct that contains 2 loxP sites I want to utilise in the future. If i combine the 2 populations of cells, will the cre being produced act on the loxP sites in the other population of cells to drop out the cassette flanked by each? If that is the case how long would this take? once i was certain the cre had been given enough time to act would i then put the cells under selection pressure to ensure a pure population of cells containing the now modified trap construct? Then extract the rna, run a pcr using primers designed for the 5' and 3' ends of the segment containing the loxP sites, run this on a gel, if i get no bands will i be able to conclude the cre works, provided i include a positive control that produces fragments using these primers? If anyone has any suggestions or modifications to this protocol i'd be very appreciative. Kind Regards Scott Brown PhD Candidate Children's Cancer Institute of Australia Support our important research into childhood cancer today! Call 1800 685 686 or visit www.ccia.org.au 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 Jul 11 04:27:40 2008 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Fri Jul 11 13:31:56 2008 Subject: stealth siRNA optimisation Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A02972A0B@mail01.ccia.local> Hi, I have been trying to use some invitrogen stealth siRNA to knock down the function of the glucocorticoid receptor and expression of BCDIN3. I am using a nucelofector for transfecting the siRNA, it recommends using 2uM [siRNA], I have tried using 5um and 10uM as well and have achieved better results (almost 80% knockdown). I always thought high concentraions of siRNA can be toxic to cells, can anyone advice on the mechanism of action responsible for this toxicity, is their any limit to how much siRNA i can expose my cell line to or is this a typical scenario when optimising siRNA knockdown. Thanks Scott Brown PhD Candidate Children's Cancer Institute of Australia Support our important research into childhood cancer today! Call 1800 685 686 or visit www.ccia.org.au 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 engelbert_buxbaum from hotmail.com Fri Jul 11 13:36:31 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri Jul 11 16:12:12 2008 Subject: IP Problem References: Message-ID: Am 10.07.2008, 13:43 Uhr, schrieb John Leonard : > Hello all, > > I'm looking to perform an IP Western of a protein that runs at around > 50-55kD on an SDS-PAGE gel - approximately the same as my antibody's > heavy > chain. As such, it could be very difficult to detect presence of my > protein This largely deppends on how you want to detect your protein after electrophoresis. Usually in IP you have too small an amount to detect by protein staining. Instead the target protein is usually metabolically labelled (originates from cell cultures grown with 35-S Met/Cys), the presence of Ig heavy chain would not interfere with detection as it is non-radioactive. A little technical hint: Protein A beads are quite expensive. For IP I use fixed Staph. aureus cells instead. These are commercially availble (e.g. Pansorbin from Calbiochem). From Sharon.Waldrop from utsouthwestern.edu Fri Jul 11 14:10:34 2008 From: Sharon.Waldrop from utsouthwestern.edu (Sharon Waldrop) Date: Fri Jul 11 16:12:17 2008 Subject: Trypsin/EDTA Message-ID: <487769DA0200003E00034ACA@swnw126.swmed.org> Hello Group, My 2 cents worth: I have different cell lines that require different methods of removing from flasks or petri dishes. Huh7 (human hepatoma): Wash with PBS, aspirate, add 0.25% Trypsin, aspirate, place in incubator for 2 minutes. Add 10 mls complete media. HTC (Rat Hepatoma): Wash with PBS, aspirate, add 0.02% EDTA - cell culture grade supplemented with 20 mls of 0.25% Trypsin, aspirate, place in incubator for 2 minutes, add complete media. HEK293 (Human embryonic): Wash with PBS (wash gently), aspirate, add 0.02% EDTA, (wash gently), aspirate, place in incubator for 1 minute, add complete media and so forth. I have quite a few cell lines - adherent and suspension. A little tedious but for what I do necessary. I hope this has helped. Good luck with your research. SLW From peter.ianakiev from gmail.com Fri Jul 11 14:50:01 2008 From: peter.ianakiev from gmail.com (peter) Date: Fri Jul 11 16:12:22 2008 Subject: Cre recombinase question References: Message-ID: <24dcbc49-58ae-406e-a55c-2350d2b03533@t54g2000hsg.googlegroups.com> On Jul 11, 5:22?am, "Scott Brown" wrote: > Hi, > > I have performed a few digests on what I am pretty certain is a Cre plasmid however none of the fragments match up to the plasmid map. > > To validate that i do infact have Cre, I was thinking of doing a cotransfection, transfecting one population of 293's with pCre and another with a trap construct that contains 2 loxP sites I want to utilise in the future. > > If i combine the 2 populations of cells, will the cre being produced act on the loxP sites in the other population of cells to drop out the cassette flanked by each? > > If that is the case how long would this take? once i was certain the cre had been given enough time to act would i then put the cells under selection pressure to ensure a pure population of cells containing the now modified trap construct? > > Then extract the rna, run a pcr using primers designed for the 5' and 3' ends of the segment containing the loxP sites, run this on a gel, if i get no bands will i be able to conclude the cre works, provided i include a positive control that produces fragments using these primers? > > If anyone has any suggestions or modifications to this protocol i'd be very appreciative. > > Kind Regards > > Scott Brown > PhD Candidate > Children's Cancer Institute of Australia > > Support our important research into childhood cancer today! Call 1800 685 686 or visitwww.ccia.org.au > > 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. Oh my, isnt is just easyer to sequence the plasmid, or PCR with cre primers? -Peter From R.Jayakumar from roswellpark.org Fri Jul 11 15:22:25 2008 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Fri Jul 11 16:12:27 2008 Subject: stealth siRNA optimisation In-Reply-To: <2A67EA781EC7F949A2AB0A0D07A86C6A02972A0B@mail01.ccia.local> References: <2A67EA781EC7F949A2AB0A0D07A86C6A02972A0B@mail01.ccia.local> Message-ID: <97101976F8A044468CA74FE11883B90E173E7ACA@VISTA.roswellpark.org> Depends a lot on the cell line and amaxa protocol. You can keep increasing it till you see an optimum between cell death and efficiency in knockdown. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Scott Brown Sent: Friday, July 11, 2008 5:28 AM To: methods@magpie.bio.indiana.edu Subject: stealth siRNA optimisation Hi, I have been trying to use some invitrogen stealth siRNA to knock down the function of the glucocorticoid receptor and expression of BCDIN3. I am using a nucelofector for transfecting the siRNA, it recommends using 2uM [siRNA], I have tried using 5um and 10uM as well and have achieved better results (almost 80% knockdown). I always thought high concentraions of siRNA can be toxic to cells, can anyone advice on the mechanism of action responsible for this toxicity, is their any limit to how much siRNA i can expose my cell line to or is this a typical scenario when optimising siRNA knockdown. Thanks Scott Brown PhD Candidate Children's Cancer Institute of Australia Support our important research into childhood cancer today! Call 1800 685 686 or visit www.ccia.org.au 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 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 R.Jayakumar from roswellpark.org Fri Jul 11 16:41:45 2008 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Fri Jul 11 20:06:56 2008 Subject: IP Problem In-Reply-To: References: Message-ID: <97101976F8A044468CA74FE11883B90E173E7ACB@VISTA.roswellpark.org> Not very difficult at all. Use a different antibody (for e.g. Mouse) for IPing the protein down and use another one for western detection (for e.g. Rabbit). Then the heavy chain of mouse will not be visible when we use anti-Rabbit secondary for western detection. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Dr Engelbert Buxbaum Sent: Friday, July 11, 2008 2:37 PM To: methods@magpie.bio.indiana.edu Subject: Re: IP Problem Am 10.07.2008, 13:43 Uhr, schrieb John Leonard : > Hello all, > > I'm looking to perform an IP Western of a protein that runs at around > 50-55kD on an SDS-PAGE gel - approximately the same as my antibody's > heavy chain. As such, it could be very difficult to detect presence > of my protein 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 stxsz1 from nottingham.ac.uk Sun Jul 13 05:37:33 2008 From: stxsz1 from nottingham.ac.uk (Zhong Silin) Date: Sun Jul 13 13:55:09 2008 Subject: detect splice variants in living cell References: <200807121704.m6CH42O26742@net.bio.net> Message-ID: Hi all How can we detect alternative splicing in a living cell? RT-PCR can be used to check alternative splicing. However, mRNA level is not always the same as protein. Promoter:reporter fusion is nearly impossible unless the reporter is in a translational fusion to the target protein. Again, it won't tell the difference of the protein splice variants. I assume that one way is to insert a fluorescent/epitope tag to a specific region of that protein, which is present/absent in its splice variants. Any suggestion please? cheers silin ________________________________ From: methods-bounces@oat.bio.indiana.edu on behalf of methods-request@oat.bio.indiana.edu Sent: Sat 7/12/2008 6:04 PM To: methods@magpie.bio.indiana.edu Subject: Methods Digest, Vol 38, 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: IP Problem (Jayakumar, R) ---------------------------------------------------------------------- Message: 1 Date: Fri, 11 Jul 2008 17:41:45 -0400 From: "Jayakumar, R" Subject: RE: IP Problem To: "Dr Engelbert Buxbaum" , Message-ID: <97101976F8A044468CA74FE11883B90E173E7ACB@VISTA.roswellpark.org> Content-Type: text/plain; charset="us-ascii" Not very difficult at all. Use a different antibody (for e.g. Mouse) for IPing the protein down and use another one for western detection (for e.g. Rabbit). Then the heavy chain of mouse will not be visible when we use anti-Rabbit secondary for western detection. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Dr Engelbert Buxbaum Sent: Friday, July 11, 2008 2:37 PM To: methods@magpie.bio.indiana.edu Subject: Re: IP Problem Am 10.07.2008, 13:43 Uhr, schrieb John Leonard : > Hello all, > > I'm looking to perform an IP Western of a protein that runs at around > 50-55kD on an SDS-PAGE gel - approximately the same as my antibody's > heavy chain. As such, it could be very difficult to detect presence > of my protein 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. ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 38, Issue 9 ************************************** 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 sudhee26 from gmail.com Sun Jul 13 12:35:00 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Sun Jul 13 13:55:20 2008 Subject: generating an empty vector! Message-ID: Hello, I have cloned my gene of interest via PCR into pcDNA3.1V5/His6 TOPO vector. However i am not very sure about the control vector. In the TOPO TA kit, an expression control vector of pcDNA3.1V5/His6 LacZ was given however i cant use if for my experiments (it does some crazy stuff :( )..i did try pcDNA3.1 without any V5 or His tag as empty vector and it seems to work fine..but just a doubt 1. should not i have have pcDNA 3.1 V5/His6 empty vector as a better control? 2. If yes then how to generate it? I guess i need not use T4 DNA ligase as TOPOisomerase itself can ligate after i use klenow 3. I beg for a Klenow protocol for this type of vector (could not find it on net) 4. Does V5/His6 tag make really big difference to the properties of a protein? if not then i can use empty pcDNA3.1 it self right? kindly suggest..in urgent need Sudheendra. -- Think before agree Think before you nod STOP thinking to be a God From pjie2 from cam.ac.uk Mon Jul 14 05:45:59 2008 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Mon Jul 14 11:26:45 2008 Subject: detect splice variants in living cell In-Reply-To: <1lvek.41$dp1.7@newsfe05.lga> References: <200807121704.m6CH42O26742@net.bio.net> <1lvek.41$dp1.7@newsfe05.lga> Message-ID: On Sun, 13 Jul 2008, DK wrote: >>"Zhong Silin" wrote: >> >> How can we detect alternative splicing in a living cell? >> >> I assume that one way is to insert a fluorescent/epitope tag to a specific >> region of that protein, which is present/absent in its splice variants. >> >> Any suggestion please? > > Why not Northern? A) Because he wants to look at protein levels, not RNA levels B) Because he wants to do it in living cells I don't see the necessity for incorporating a reporter tag into the gene - would it not be simpler to raise an antibody specifically to the variant splice isoform? Introducing a tag would seem to run a large risk of altering the function of the tagged exon. Peter From pjie2 from cam.ac.uk Mon Jul 14 09:28:05 2008 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Mon Jul 14 11:26:50 2008 Subject: detect splice variants in living cell In-Reply-To: References: <200807121704.m6CH42O26742@net.bio.net> <1lvek.41$dp1.7@newsfe05.lga> Message-ID: On Mon, 14 Jul 2008, DK wrote: > Peter Ellis wrote: >> >> I don't see the necessity for incorporating a reporter tag into the gene - >> would it not be simpler to raise an antibody specifically to the variant >> splice isoform? > > That's not going to be quantitative. Any two polyclonals > are going to give different results. Not sure the OP was asking for something quantitative. My reading is that he wants some way to label the particular splice variants so that they can be distinguished from each other within a living cell - perhaps to check if there are subcellular localisation differences between variants? It would help if we knew why the cells need to be living rather than fixed. Peter From k.schink_nospam_ from _nospam_gmail.com Mon Jul 14 10:07:57 2008 From: k.schink_nospam_ from _nospam_gmail.com (Kay Schink) Date: Mon Jul 14 11:26:55 2008 Subject: generating an empty vector! In-Reply-To: References: Message-ID: Sudheendra Rao N R schrieb: > Hello, > I have cloned my gene of interest via PCR into pcDNA3.1V5/His6 TOPO vector. > However i am not very sure about the control vector. In the TOPO TA kit, an > expression control vector of pcDNA3.1V5/His6 LacZ was given however i cant > use if for my experiments (it does some crazy stuff :( )..i did try pcDNA3.1 > without any V5 or His tag as empty vector and it seems to work fine..but > just a doubt > 1. should not i have have pcDNA 3.1 V5/His6 empty vector as a better > control? > 2. If yes then how to generate it? I guess i need not use T4 DNA ligase as > TOPOisomerase itself can ligate after i use klenow > 3. I beg for a Klenow protocol for this type of vector (could not find it on > net) > 4. Does V5/His6 tag make really big difference to the properties of a > protein? if not then i can use empty pcDNA3.1 it self right? > > kindly suggest..in urgent need > > Sudheendra. > How about using a vector obtained from a negative clone... If you do a TOPO cloning reaction, you usually get some clones that have an empty, self-ligated vector (at least with our Topo cloning kit you get it quite frequently ;-) ) These vectors should be empty and be a suitable control.. Good luck Kay From usiva from ufl.edu Mon Jul 14 11:46:12 2008 From: usiva from ufl.edu (SIVAKUMAR) Date: Mon Jul 14 13:37:33 2008 Subject: enzyme activity calculation-clarification Message-ID: <1675639986.125741216053972207.JavaMail.osg@osgjas02.cns.ufl.edu> Hi Does any one could clarify the correctness of the enzyme activity unit calculation? ???526/min x Reaction vol. Enzyem activity (U / ml)= ____________________________ x dilution factor 0.065x Enzyme vol. For e.g., 0.03x0.2 _________x5# = 9.2 U /ml or 153.36 nanokatals/ml 0.065x0.05 where, Change in abs at 526 per min was 0.03 Assay volume 0.2 ml Enzyme volume 0.05 ml Extn coeff: = 65,000 /M/cm = 65 /Mm/cm = 0.065 /uM/cm* * used 0.065, since EU is defined u mole of substrate oxidized. 5# since the assay volume was 0.2ml, I multiplied the whole by 5 to get per ml value Is it correct? From pow.joshi from gmail.com Mon Jul 14 12:09:37 2008 From: pow.joshi from gmail.com (Pow Joshi) Date: Mon Jul 14 13:37:39 2008 Subject: generating an empty vector! In-Reply-To: References: Message-ID: <710764ea0807141009v66377786r9c8f5099d95e32d7@mail.gmail.com> 2008/7/14 Kay Schink : > Sudheendra Rao N R schrieb: > >> Hello, >> I have cloned my gene of interest via PCR into pcDNA3.1V5/His6 TOPO >> vector. >> However i am not very sure about the control vector. In the TOPO TA kit, >> an >> expression control vector of pcDNA3.1V5/His6 LacZ was given however i cant >> use if for my experiments (it does some crazy stuff :( )..i did try >> pcDNA3.1 >> without any V5 or His tag as empty vector and it seems to work fine..but >> just a doubt >> 1. should not i have have pcDNA 3.1 V5/His6 empty vector as a better >> control? >> 2. If yes then how to generate it? I guess i need not use T4 DNA ligase as >> TOPOisomerase itself can ligate after i use klenow >> 3. I beg for a Klenow protocol for this type of vector (could not find it >> on >> net) >> 4. Does V5/His6 tag make really big difference to the properties of a >> protein? if not then i can use empty pcDNA3.1 it self right? >> >> kindly suggest..in urgent need >> >> Sudheendra. >> >> How about using a vector obtained from a negative clone... If you do a > TOPO cloning reaction, you usually get some clones that have an empty, > self-ligated vector (at least with our Topo cloning kit you get it quite > frequently ;-) ) These vectors should be empty and be a suitable control.. > > Good luck yes, I used to get large amounts of empty vectors as well.... self-ligated....and we did use them as controls. Pow > > > Kay > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From ucgatan from ucl.ac.uk Mon Jul 14 12:57:41 2008 From: ucgatan from ucl.ac.uk (Tom Anderson) Date: Mon Jul 14 13:37:46 2008 Subject: detect splice variants in living cell In-Reply-To: References: <200807121704.m6CH42O26742@net.bio.net> <1lvek.41$dp1.7@newsfe05.lga> Message-ID: On Mon, 14 Jul 2008, Peter Ellis wrote: > On Sun, 13 Jul 2008, DK wrote: > >>"Zhong Silin" wrote: > >> > >> How can we detect alternative splicing in a living cell? > >> > >> I assume that one way is to insert a fluorescent/epitope tag to a specific > >> region of that protein, which is present/absent in its splice variants. > >> > >> Any suggestion please? > > > > Why not Northern? > > A) Because he wants to look at protein levels, not RNA levels > > B) Because he wants to do it in living cells > > I don't see the necessity for incorporating a reporter tag into the gene > - would it not be simpler to raise an antibody specifically to the > variant splice isoform? Raising antibodies is not exactly trivial. Particularly with that degree of specificity - if the alternation includes or excludes a big chunk of protein, you just make an antibody against that, but if it's just a few residues, you're in trouble. > Introducing a tag would seem to run a large risk of altering the > function of the tagged exon. Yes. This might not matter, though. It might also affect the splicing itself, which would matter. Not that i have a better solution! A particular challenge is looking at a protein with multiple alternative splicing sites. I was looking at tropomyosin a couple of years ago: four genes, each with splicing at two sites (or three? i forget). I wanted to know (dead cells would be fine, in my case), how the pools of A1a, A1b, A2a, A2b, B1a, B1b, etc compared. I never came up with a good solution. 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 joseolucha from gmail.com Mon Jul 14 16:27:40 2008 From: joseolucha from gmail.com (Jose Olucha) Date: Mon Jul 14 17:13:45 2008 Subject: Looking for a protocol to obtain N10-Formyl THF Message-ID: Dear All, I am looking for a protocol to be able to make and purify N10-formyl tetrahydrofolate to use as a (suspected) co-factor for a transformylase. So far I have yet to find a protocol in literature where the author actually made and purified N10-f-THF, maybe I am not looking in the right direction? I've been using Google scholar to no avail. Any hints of a protocol or a paper that might help? Thanks! Jose From pow.joshi from gmail.com Mon Jul 14 18:46:31 2008 From: pow.joshi from gmail.com (Pow Joshi) Date: Mon Jul 14 21:07:21 2008 Subject: generating an empty vector! In-Reply-To: <710764ea0807141645p95a2937sad9d7ba3fd26a1be@mail.gmail.com> References: <710764ea0807141645p95a2937sad9d7ba3fd26a1be@mail.gmail.com> Message-ID: <710764ea0807141646o6c35742i840f967a6e425f40@mail.gmail.com> . 2008/7/14 Pow Joshi : > > > 2008/7/14 DK : > > In article , "Pow Joshi" >> wrote: >> >2008/7/14 Kay Schink : >> > >> >> Sudheendra Rao N R schrieb: >> >> >> >>> Hello, >> >>> I have cloned my gene of interest via PCR into pcDNA3.1V5/His6 TOPO >> >>> vector. >> >>> However i am not very sure about the control vector. In the TOPO TA >> kit, >> >>> an >> >>> expression control vector of pcDNA3.1V5/His6 LacZ was given however i >> cant >> >>> use if for my experiments (it does some crazy stuff :( )..i did try >> >>> pcDNA3.1 >> >>> without any V5 or His tag as empty vector and it seems to work >> fine..but >> >>> just a doubt >> >>> 1. should not i have have pcDNA 3.1 V5/His6 empty vector as a better >> >>> control? >> >>> 2. If yes then how to generate it? I guess i need not use T4 DNA >> ligase as >> >>> TOPOisomerase itself can ligate after i use klenow >> >>> 3. I beg for a Klenow protocol for this type of vector (could not find >> it >> >>> on >> >>> net) >> >>> 4. Does V5/His6 tag make really big difference to the properties of a >> >>> protein? if not then i can use empty pcDNA3.1 it self right? >> >>> >> >>> kindly suggest..in urgent need >> >>> >> >>> Sudheendra. >> >>> >> >>> How about using a vector obtained from a negative clone... If you do >> a >> >> TOPO cloning reaction, you usually get some clones that have an empty, >> >> self-ligated vector (at least with our Topo cloning kit you get it >> quite >> >> frequently ;-) ) These vectors should be empty and be a suitable >> control.. >> >> >> >> Good luck >> > >> > >> >yes, I used to get large amounts of empty vectors as well.... >> >self-ligated....and we did use them as controls. >> >> Poor control then. As I mention in another post, such a vector >> will not be expressing any protein with a 45 aa peptide that might >> very well affect results. E.g: >> >> http://www.ncbi.nlm.nih.gov/pubmed/11244567 >> >> "The fusion of these epitopes with the recombinant proteins is not >> expected to alter the behavior of the protein of interest. In this report, >> we demonstrate that the mere expression of a cellular protein, >> hVIP/mov34, which we earlier identified as a cellular HIV-1 Vpr >> ligand, in two different vectors clearly altered its localization pattern >> in HeLa cells. Specifically, cloning of hVIP/mov34 in pcDNA3/HisA >> resulted in its nuclear localization, whereas the expression of this >> gene from a TOPO cloning expression vector, pcDNA3.1/V5/His, >> resulted in cytoplasmic expression. The native staining pattern >> of hVIP/mov34 using polyclonal antisera raised against hVIP/mov34 >> demonstrated cytoplasmic staining. During cloning, other leader >> sequences intended for targeting this protein into a cytoplasmic >> or a nuclear location were not fused to the actual ORF of this >> protein. Also, the amino acid sequence of the fusion region arising >> from cloning of hVIP/mov34 in both vectors does not match >> any reported NLS sequences. These results indicate that the >> choice of the expression vectors, as well as the position of >> synthetic epitopes, can significantly alter the behavior and >> the biology of recombinant proteins. This result suggests the >> need for a careful examination of these features when >> characterizing a newly identified protein." > > > WOW... thanks Dima .... Frankly, never bothered about it since we were > doing some specific IPs.... it seemed to work.... I see though that it is > definitely a concern. > > Pow > >> >> >> DK >> _______________________________________________ >> Methods mailing list >> Methods@net.bio.net >> http://www.bio.net/biomail/listinfo/methods >> > > From sudhee26 from gmail.com Mon Jul 14 23:15:34 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Tue Jul 15 07:41:27 2008 Subject: generating an empty vector! In-Reply-To: <710764ea0807141646o6c35742i840f967a6e425f40@mail.gmail.com> References: <710764ea0807141645p95a2937sad9d7ba3fd26a1be@mail.gmail.com> <710764ea0807141646o6c35742i840f967a6e425f40@mail.gmail.com> Message-ID: Hello, as far as remember, pcDNA3.1 does have a putative transcription start..wont that make sure that tags are expressed even in empty vector which has got self ligated..has any one tried detected overexpressed empty vector products in a blot using V5 or His antibody? On Tue, Jul 15, 2008 at 5:16 AM, Pow Joshi wrote: > . > > 2008/7/14 Pow Joshi : > > > > > > > 2008/7/14 DK : > > > > In article , "Pow > Joshi" > >> wrote: > >> >2008/7/14 Kay Schink : > >> > > >> >> Sudheendra Rao N R schrieb: > >> >> > >> >>> Hello, > >> >>> I have cloned my gene of interest via PCR into pcDNA3.1V5/His6 TOPO > >> >>> vector. > >> >>> However i am not very sure about the control vector. In the TOPO TA > >> kit, > >> >>> an > >> >>> expression control vector of pcDNA3.1V5/His6 LacZ was given however > i > >> cant > >> >>> use if for my experiments (it does some crazy stuff :( )..i did try > >> >>> pcDNA3.1 > >> >>> without any V5 or His tag as empty vector and it seems to work > >> fine..but > >> >>> just a doubt > >> >>> 1. should not i have have pcDNA 3.1 V5/His6 empty vector as a better > >> >>> control? > >> >>> 2. If yes then how to generate it? I guess i need not use T4 DNA > >> ligase as > >> >>> TOPOisomerase itself can ligate after i use klenow > >> >>> 3. I beg for a Klenow protocol for this type of vector (could not > find > >> it > >> >>> on > >> >>> net) > >> >>> 4. Does V5/His6 tag make really big difference to the properties of > a > >> >>> protein? if not then i can use empty pcDNA3.1 it self right? > >> >>> > >> >>> kindly suggest..in urgent need > >> >>> > >> >>> Sudheendra. > >> >>> > >> >>> How about using a vector obtained from a negative clone... If you > do > >> a > >> >> TOPO cloning reaction, you usually get some clones that have an > empty, > >> >> self-ligated vector (at least with our Topo cloning kit you get it > >> quite > >> >> frequently ;-) ) These vectors should be empty and be a suitable > >> control.. > >> >> > >> >> Good luck > >> > > >> > > >> >yes, I used to get large amounts of empty vectors as well.... > >> >self-ligated....and we did use them as controls. > >> > >> Poor control then. As I mention in another post, such a vector > >> will not be expressing any protein with a 45 aa peptide that might > >> very well affect results. E.g: > >> > >> http://www.ncbi.nlm.nih.gov/pubmed/11244567 > >> > >> "The fusion of these epitopes with the recombinant proteins is not > >> expected to alter the behavior of the protein of interest. In this > report, > >> we demonstrate that the mere expression of a cellular protein, > >> hVIP/mov34, which we earlier identified as a cellular HIV-1 Vpr > >> ligand, in two different vectors clearly altered its localization > pattern > >> in HeLa cells. Specifically, cloning of hVIP/mov34 in pcDNA3/HisA > >> resulted in its nuclear localization, whereas the expression of this > >> gene from a TOPO cloning expression vector, pcDNA3.1/V5/His, > >> resulted in cytoplasmic expression. The native staining pattern > >> of hVIP/mov34 using polyclonal antisera raised against hVIP/mov34 > >> demonstrated cytoplasmic staining. During cloning, other leader > >> sequences intended for targeting this protein into a cytoplasmic > >> or a nuclear location were not fused to the actual ORF of this > >> protein. Also, the amino acid sequence of the fusion region arising > >> from cloning of hVIP/mov34 in both vectors does not match > >> any reported NLS sequences. These results indicate that the > >> choice of the expression vectors, as well as the position of > >> synthetic epitopes, can significantly alter the behavior and > >> the biology of recombinant proteins. This result suggests the > >> need for a careful examination of these features when > >> characterizing a newly identified protein." > > > > > > WOW... thanks Dima .... Frankly, never bothered about it since we were > > doing some specific IPs.... it seemed to work.... I see though that it is > > definitely a concern. > > > > Pow > > > >> > >> > >> DK > >> _______________________________________________ > >> 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 > -- Think before agree Think before you nod STOP thinking to be a God From usiva from ufl.edu Tue Jul 15 09:59:47 2008 From: usiva from ufl.edu (SIVAKUMAR) Date: Tue Jul 15 13:12:35 2008 Subject: enzyme activity calculation-clarification Message-ID: <524429832.175981216133987567.JavaMail.osg@osgjas02.cns.ufl.edu> Hi Does any one could clarify the correctness of the enzyme activity unit calculation? 526/min x Reaction vol. Enzyem activity(U/ml)=______________________x dilution factor 0.065x Enzyme vol. For e.g., 0.03x0.2 _________x5# = 9.2 U /ml or 153.36 nanokatals/ml 0.065x0.05 where, Change in abs at 526 per min was 0.03 Assay volume 0.2 ml Enzyme volume 0.05 ml Extn coeff: = 65,000 /M/cm = 65 /Mm/cm = 0.065 /uM/cm* * used 0.065, since EU is defined u mole of substrate oxidized. 5# since the assay volume was 0.2ml, I multiplied the whole by 5 to get per ml value Is it correct? Siva From sudhee26 from gmail.com Tue Jul 15 14:14:07 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Tue Jul 15 18:23:27 2008 Subject: generating an empty vector! In-Reply-To: References: <710764ea0807141645p95a2937sad9d7ba3fd26a1be@mail.gmail.com> <710764ea0807141646o6c35742i840f967a6e425f40@mail.gmail.com> Message-ID: Hi DK, I agree. common Kozak being (G/A)NNATGG Whats that "putative transcriptional start" after 3' end of CMV promoter? http://tools.invitrogen.com/content/sfs/vectors/pcdna3.1v5histopo_mcs.pdf TAG is amber codon..i dont get this!! Sudheendra. On Tue, Jul 15, 2008 at 7:04 PM, DK wrote: > In article , "Sudheendra > Rao N R" wrote: > >Hello, > >as far as remember, pcDNA3.1 does have a putative transcription > start..wont > >that make sure that tags are expressed even in empty vector which has got > >self ligated.. > > No, it won't. The plasmid lacks *translation* start (that needs to be > supplied with a cloned gene). > > http://tools.invitrogen.com/content/sfs/vectors/pcdna3.1v5histopo_mcs.pdf > > Off topic note: > Which means that if only ORF, without any extra sequences is > cloned, the immediate sequence upstream of the AUG will be > GCCCTT - don't think it it fits Kozak consensus very well. So, > likely relatively poor expression levels can be expected. > > DK > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod STOP thinking to be a God From vadivelarunachalam from yahoo.com Wed Jul 16 05:16:27 2008 From: vadivelarunachalam from yahoo.com (V Arunachalam) Date: Wed Jul 16 08:44:06 2008 Subject: Anyone has tried a simple vector for Agro mediated Arabidopsis Message-ID: <862172.60528.qm@web94712.mail.in2.yahoo.com> Hi Is it right to use a simple cloning vector for Agrobacterium mediated floral?dip transformation to verify promoter strength by transient expression. Has anyone tried it. Pl reply ?With best wishes, V.Arunachalam Sr Scientist Horticulture Genetics lab. CPCRI Kasaragod 671 124 Kerala DBT Postdoctoral Fellow Genetic transformation Lab. ICRISAT Patancheru 502324 AP India E-mail : vadivelarunachalam@yahoo.com Unlimited freedom, unlimited storage. Get it now, on http://help.yahoo.com/l/in/yahoo/mail/yahoomail/tools/tools-08.html/ From mprigge from indiana.edu Wed Jul 16 12:21:34 2008 From: mprigge from indiana.edu (Michael J. Prigge) Date: Wed Jul 16 13:16:42 2008 Subject: Anyone has tried a simple vector for Agro mediated Arabidopsis Message-ID: pBluescript, pUC, pGEM, etc. will not work directly, of course, as they lack T-DNA border(s) and Agro-compatible origins of replication. Being a postdoc from a transformation lab, I'm assuming that you'd know that though. Are you wondering if you can clone these things into pBluescript, pUC, etc. along with the promoter:reporter? If so, you probably could, but it'd be a lot easier (and more easily comparable to others' data) to use any one of the multitude of existing vectors. Mike > Hi > Is it right to use a simple cloning vector for Agrobacterium > mediated floral dip transformation to verify promoter strength by > transient expression. Has anyone tried it. Pl reply > With best wishes, > V.Arunachalam > Sr Scientist Horticulture > Genetics lab. CPCRI Kasaragod 671 124 Kerala > DBT Postdoctoral Fellow Genetic transformation Lab. > ICRISAT Patancheru 502324 AP > India From tracyscience from gmail.com Wed Jul 16 15:39:55 2008 From: tracyscience from gmail.com (TC) Date: Wed Jul 16 19:21:24 2008 Subject: DNA crosslinking on nylon memberane Message-ID: <2387671c0807161339j146896e1q62cfce5df96d9946@mail.gmail.com> Hello, I am trying to crosslink DNA to nylon membrane for southern blots. Besides traditional UV crosslinking (may induce DNA damage) and baking (causes DNA to denature), are there any other way to crosslink DNA onto nylon memberanes? For my purpose, I hope minimize DNA damage and keep that DNA in native condition for the Southern blots. THANKS~! Tracy From csibapriya from yahoo.co.in Wed Jul 16 13:46:40 2008 From: csibapriya from yahoo.co.in (Sibapriya Chaudhuri) Date: Wed Jul 16 19:21:30 2008 Subject: cloning in duet vector Message-ID: <493575.29911.qm@web8507.mail.in.yahoo.com> hi i m tryin to clone 2 genes in pRSF duet vector. the first gene has been successfully cloned and even its expression level checked by induction. i was able to effectively screen the first clone bur somehow i am unable to clone the second gene. I am restricting the and mcs wit 2 different enzymes-hence not using phosphatase. the vector is being digested properly as evident from the gel profile. but not gettin nay positive clones while tryin to screen the transformants after ligation can anyone help? Explore your hobbies and interests. Go to http://in.promos.yahoo.com/groups/ From tracyscience from gmail.com Wed Jul 16 15:42:30 2008 From: tracyscience from gmail.com (TC) Date: Wed Jul 16 19:21:35 2008 Subject: DNA crosslinking on nylon membrane Message-ID: <2387671c0807161342s2e9375fek3ac7958fed85bf4b@mail.gmail.com> Hello, I am trying to crosslink DNA to nylon membrane for southern blots. Besides traditional UV crosslinking (may induce DNA damage) and baking (causes DNA to denature), are there any other ways to crosslink DNA onto nylon memberanes? For my purpose, I hope minimize DNA damage and keep that DNA in native condition for the Southern blots. THANKS~! Tracy From rory.obrien from stonebow.otago.ac.nz Wed Jul 16 19:34:08 2008 From: rory.obrien from stonebow.otago.ac.nz (Rory O'Brien) Date: Wed Jul 16 21:44:44 2008 Subject: DNA crosslinking on nylon memberane In-Reply-To: <2387671c0807161339j146896e1q62cfce5df96d9946@mail.gmail.com> Message-ID: Hi Tracy, I think DNA binds pretty strongly to positively charged membranes. I seem to recall several occasions where, using charged membranes, crosslinking was omitted but didn't seem to make any discernable difference... HTH, - Rory > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu > [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of TC > Sent: 17 July 2008 08:40 > To: methods@magpie.bio.indiana.edu > Subject: DNA crosslinking on nylon memberane > > Hello, > > I am trying to crosslink DNA to nylon membrane for southern > blots. Besides traditional UV crosslinking (may induce DNA > damage) and baking (causes DNA to denature), are there any > other way to crosslink DNA onto nylon memberanes? For my > purpose, I hope minimize DNA damage and keep that DNA in > native condition for the Southern blots. > > THANKS~! > > Tracy > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods From pjie2 from cam.ac.uk Thu Jul 17 03:49:26 2008 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Thu Jul 17 12:21:03 2008 Subject: DNA crosslinking on nylon memberane In-Reply-To: References: Message-ID: <6e8fcoF5s560U1@mid.individual.net> TC wrote: > Hello, > > I am trying to crosslink DNA to nylon membrane for southern blots. Besides > traditional UV crosslinking (may induce DNA damage) and baking (causes DNA > to denature), are there any other way to crosslink DNA onto nylon > memberanes? For my purpose, I hope minimize DNA damage and keep that DNA in > native condition for the Southern blots. Why do you want your DNA in native condition? Unless you denature it, it won't be accessible for your probe to bid to and you will have reduced signal. Peter From josenet from tiscali.co.uk Thu Jul 17 04:14:26 2008 From: josenet from tiscali.co.uk (Jose de las Heras) Date: Thu Jul 17 12:21:11 2008 Subject: DNA crosslinking on nylon memberane References: Message-ID: <6e8grtF5j9udU1@mid.individual.net> "TC" wrote in message news:mailman.777.1216254116.3533.methods@net.bio.net... > Hello, > > I am trying to crosslink DNA to nylon membrane for southern blots. > Besides > traditional UV crosslinking (may induce DNA damage) and baking (causes DNA > to denature), are there any other way to crosslink DNA onto nylon > memberanes? For my purpose, I hope minimize DNA damage and keep that DNA > in > native condition for the Southern blots. > > THANKS~! > > Tracy Several positively charged membranes claimed to be ok for use without conventional crosslinking. Personally I always UV-crosslink out of habit. I doubt the damage on immobilised DNA is going to give you any trouble, and I doubt immobilised DNA on a dry membrane is going to denature with heat... and besides, for successful hybridisation you want the DNA to be denatured. You may try a positively charged membrane to bypass heating/UV. Out of curiosity, what are you trying to do? Jose From bt8a002 from uni-hamburg.de Thu Jul 17 06:44:56 2008 From: bt8a002 from uni-hamburg.de (Peter Willingmann) Date: Thu Jul 17 12:21:17 2008 Subject: Homemade FTA paper and kit??? Message-ID: <6e8pa2F5etbmU1@mid.dfncis.de> hello netters; can anybody point me to a paper where the basis of FTA techniques from Whatman is described in some more detail?? I would like to have a better understanding how this work and perhaps to make a homemade kit. thanks in advance ciao peter From ttchow from gmail.com Thu Jul 17 11:04:33 2008 From: ttchow from gmail.com (Tracy Chow) Date: Thu Jul 17 12:21:31 2008 Subject: DNA crosslinking on nylon memberane In-Reply-To: References: <2387671c0807161339j146896e1q62cfce5df96d9946@mail.gmail.com> Message-ID: <7110c53e0807170904o64fe9694j7e7978a02921a03b@mail.gmail.com> Hi Rory, Thanks for your suggestion...really appreciate. May I ask if you use Hybond-N+ or Hybond-XL (also positively charged) membrane? May I ask for the conditions you dry the membrane (room temperature or vacuum)? How long do you *dry the membranes *prior to hybridization? How long do you *pre-hybridize* prior to probe incubation? THANKS!!! Tracy On Wed, Jul 16, 2008 at 7:34 PM, Rory O'Brien < rory.obrien@stonebow.otago.ac.nz> wrote: > Hi Tracy, > > I think DNA binds pretty strongly to positively charged membranes. I seem > to > recall several occasions where, using charged membranes, crosslinking was > omitted but didn't seem to make any discernable difference... > > HTH, > > - Rory > > > > -----Original Message----- > > From: methods-bounces@oat.bio.indiana.edu > > [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of TC > > Sent: 17 July 2008 08:40 > > To: methods@magpie.bio.indiana.edu > > Subject: DNA crosslinking on nylon memberane > > > > Hello, > > > > I am trying to crosslink DNA to nylon membrane for southern > > blots. Besides traditional UV crosslinking (may induce DNA > > damage) and baking (causes DNA to denature), are there any > > other way to crosslink DNA onto nylon memberanes? For my > > purpose, I hope minimize DNA damage and keep that DNA in > > native condition for the Southern blots. > > > > THANKS~! > > > > Tracy > > _______________________________________________ > > 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 > From tracyscience from gmail.com Thu Jul 17 13:39:49 2008 From: tracyscience from gmail.com (TC) Date: Thu Jul 17 16:11:09 2008 Subject: DNA crosslinking on nylon memberane In-Reply-To: <7110c53e0807171125q4925decar596e9d4b26a653aa@mail.gmail.com> References: <6e8grtF5j9udU1@mid.individual.net> <7110c53e0807171125q4925decar596e9d4b26a653aa@mail.gmail.com> Message-ID: <2387671c0807171139l26e77025oacd570a89811f034@mail.gmail.com> Hi all, Thanks for sharing your thoughts. I hope to study the overhangs of the DNA; therefore, need to keep DNA in native conditions. Tracy On Thu, Jul 17, 2008 at 1:25 PM, Tracy Chow wrote: > > > ---------- Forwarded message ---------- > From: Jose de las Heras > Date: Thu, Jul 17, 2008 at 4:14 AM > Subject: Re: DNA crosslinking on nylon memberane > To: methods@magpie.bio.indiana.edu > > > > "TC" wrote in message > news:mailman.777.1216254116.3533.methods@net.bio.net... > > Hello, > > > > I am trying to crosslink DNA to nylon membrane for southern blots. > > Besides > > traditional UV crosslinking (may induce DNA damage) and baking (causes > DNA > > to denature), are there any other way to crosslink DNA onto nylon > > memberanes? For my purpose, I hope minimize DNA damage and keep that DNA > > in > > native condition for the Southern blots. > > > > THANKS~! > > > > Tracy > > Several positively charged membranes claimed to be ok for use without > conventional crosslinking. Personally I always UV-crosslink out of habit. > > I doubt the damage on immobilised DNA is going to give you any trouble, and > I doubt immobilised DNA on a dry membrane is going to denature with heat... > and besides, for successful hybridisation you want the DNA to be denatured. > > You may try a positively charged membrane to bypass heating/UV. > > Out of curiosity, what are you trying to do? > > Jose > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > From Sharon.Waldrop from utsouthwestern.edu Thu Jul 17 15:51:01 2008 From: Sharon.Waldrop from utsouthwestern.edu (Sharon Waldrop) Date: Thu Jul 17 16:11:22 2008 Subject: Qiagen LyseBlue Message-ID: <487F6A660200003E000351B6@swnw126.swmed.org> Hello Group, Does anyone know what components are in LyseBlue? Do you, perchance, know the formula? SLW From bt8a002 from uni-hamburg.de Fri Jul 18 04:01:18 2008 From: bt8a002 from uni-hamburg.de (Peter Willingmann) Date: Fri Jul 18 07:59:32 2008 Subject: Homemade FTA paper and kit??? In-Reply-To: References: <6e8pa2F5etbmU1@mid.dfncis.de> Message-ID: <6eb437F670c9U1@mid.dfncis.de> DK wrote: thanks a lot... how stupid of me not to come to the same conclusion.. ciao peter > > Nothing fancy there. It's just a thick filter paper with dried Tris, > EDTA and SDS. You spot cell onto it, SDS lyses them, then > the whole thing is dried. Quick wash with TE removes some > DNA and most of the SDS/EDTA, then more DNA is eluted > and used for transformation (when storing clones) or PCR. > For cleaner things, phenol and/or isopropanol washes > can be added before elution. > > It is described in the multitude of patents by inventor > Leigh A. Burgoyne and Assignees either Flinders Technologies > or Whatman. E.g., # 5496562: > > ~ 50 ul of 2% SDS, 10 mM EDTA, 60 mM tris spotted on > 1 cm2 of Whatman 3MM and dried. > > FTA kits have some other filter paper, thicker than 3MM but > it really does not matter. Also, the concentrations seem to > be an overkill. Long ago, I soaked some 3 MM in just > TE + 1% SDS, dried and spotted 5 ul of several overnight > cultures. A year after, storing the paper in the drawer, all > clones gave lots of transformants after electroporation. > > This is a good candidate for inclusion into lab generics Wiki > found at http://methodsandreagents.pbwiki.com/ > > DK From chemdude321 from gmail.com Fri Jul 18 09:53:47 2008 From: chemdude321 from gmail.com (Josh Levin) Date: Fri Jul 18 12:01:22 2008 Subject: research paper required urgently References: <08c4dabc-03e4-45c7-aa16-eaac5a8ccb24@r66g2000hsg.googlegroups.com> Message-ID: On Jul 17, 7:29?am, sheelu wrote: > Dear friends, > > I need a papers published in journal "BIOTECHNOLOGY PROGRESS" entitled > "Process Design and Costing of Bioethanol Technology: A Tool for > Determining the Status and Direction of Research and Development" by > Robert Wooley, Mark Ruth, David Glassner, and John Sheehan in 1999 vol > 15 issue 5(794-803) and link of which ishttps://pubs.acs.org/secure/login?url=http%3A%2F%2Fpubs.acs.org%2Fcgi.... > As this is of great importance to me, but unable to access it, if u > can access it please send it to me. > > thanx in advance Dear Sheelu, I would recommend directly emailing the corresponding author. The authors are usually happy to provide you with a PDF reprint of the article. Josh From justin from baiardo.net Fri Jul 18 09:57:27 2008 From: justin from baiardo.net (Justin Baiardo) Date: Fri Jul 18 12:01:34 2008 Subject: Protocol for identification of unknown bacteria Message-ID: <200807181457.m6IEvaO26491@net.bio.net> Mr. Coutts- My name is Justin Baiardo and I teach microbiology at a high school out here in New Mexico. I read your post on bio.net regarding a protocol for bacterial identification through 16S PCR and then electrophoresis to compare banding patterns of 'unknowns' that i will supply the kids to knowns that we have in the lab. 1. I would like a protocol for how to do this. I have heard that the haeIII restriction enz is a good one to use. 2. I dont know how to get 16s primers. I've done searches on the internet and I cant seem to find them. I have heard that the BSF-343 and BMB-C are good ones to use. 3. I dont need to sequences, just PCR, restriction digest, then run the gels. Any help would be appreciated. Thanks- Justin Baiardo From legatek from hotmail.com Fri Jul 18 13:36:36 2008 From: legatek from hotmail.com (Kyle Legate) Date: Fri Jul 18 14:34:55 2008 Subject: DNA crosslinking on nylon memberane In-Reply-To: <6e8fcoF5s560U1@mid.individual.net> References: <6e8fcoF5s560U1@mid.individual.net> Message-ID: <6ec656F6fq6pU2@mid.individual.net> Peter Ellis wrote: > TC wrote: >> Hello, >> >> I am trying to crosslink DNA to nylon membrane for southern blots. >> Besides >> traditional UV crosslinking (may induce DNA damage) and baking (causes >> DNA >> to denature), are there any other way to crosslink DNA onto nylon >> memberanes? For my purpose, I hope minimize DNA damage and keep that >> DNA in >> native condition for the Southern blots. > > Why do you want your DNA in native condition? Unless you denature it, > it won't be accessible for your probe to bid to and you will have > reduced signal. > Not to mention that the type of DNA damage induced by UV does not inhibit hybridization efficiency one bit. Whoops, I mentioned it. UV crosslink the nylon filter, bake the nitrocellulose filter. That's how it's done. From legatek from hotmail.com Fri Jul 18 13:45:22 2008 From: legatek from hotmail.com (Kyle Legate) Date: Fri Jul 18 14:35:01 2008 Subject: DNA crosslinking on nylon memberane In-Reply-To: References: <2387671c0807161339j146896e1q62cfce5df96d9946@mail.gmail.com> Message-ID: <6ec6lkF618tlU1@mid.individual.net> Tracy Chow wrote: > Hi Rory, > > Thanks for your suggestion...really appreciate. > > May I ask if you use Hybond-N+ or Hybond-XL (also positively charged) > membrane? May I ask for the conditions you dry the membrane (room > temperature or vacuum)? How long do you *dry the membranes *prior to > hybridization? How long do you *pre-hybridize* prior to probe incubation? > > THANKS!!! > > Tracy > I'm not Rory, but I'll toss in my 0.5 cents anyway. Both membranes will be fine, but you crosslink them differently. N+ is nitrocellulose, XL is nylon, so heat or UV, respectively. Dry the membrane on some filter paper or other uncontaminated holding device by RT evaporation until there's no visible moistness before proceeding. The time it takes to clean up the blotting set-up should be enough time. If protected in a plastic sleeve or a book they can be stored dry for months before hybridizing, or they can be used immediately after crosslinking (~10 minutes after obtaining them). I used Church buffer for a pre-hyb solution and 1 hour in a hybridization oven (65C) was sufficient. Enough time to label your probe. From vivekdna from gmail.com Fri Jul 18 16:11:50 2008 From: vivekdna from gmail.com (vivekdna@gmail.com) Date: Fri Jul 18 17:47:21 2008 Subject: Homemade FTA paper and kit??? References: <6e8pa2F5etbmU1@mid.dfncis.de> Message-ID: <14a2989f-c0fb-4397-bc3d-52de8faa4a06@p25g2000hsf.googlegroups.com> Great thread. The patent mentions adding Uric Acid for ling term storage tetracetic acid and iii an anionic detergent such as sulphate and optionally iv uric acid or a urate salt By way of example a particularly according to this aspect of the absorbent cellulose based paper such a minimal loading per sq cm of a EDTA 0.5 micromols 146.1 mg b Tris 8 micromols 968.8 mg of c SDS 1 mg and optionally
Solid medium and method for DNA storage Leigh A. Burgoyne VK On Jul 17, 8:13?am, d...@no.email.thankstospam.net (DK) wrote: > In article <6e8pa2F5etb...@mid.dfncis.de>, Peter Willingmann wrote: > >hello netters; > >can anybody point me to a paper where the basis of FTA techniques from > >Whatman is described in some more detail?? > >I would like to have a better understanding how this work and perhaps > >to make a homemade kit. > > Nothing fancy there. It's just a thick filter paper with dried Tris, > EDTA and SDS. You spot cell onto it, SDS lyses them, then > the whole thing is dried. Quick wash with TE removes some > DNA and most of the SDS/EDTA, then more DNA is eluted > and used for transformation (when storing clones) or PCR. > For cleaner things, phenol and/or isopropanol washes > can be added before elution. > > It is described in the multitude of patents by inventor > Leigh A. Burgoyne and Assignees either Flinders Technologies > or Whatman. E.g., # 5496562: > > ~ 50 ul of 2% SDS, 10 mM EDTA, 60 mM tris spotted on > 1 cm2 of Whatman 3MM and dried. > > FTA kits have some other filter paper, thicker than 3MM but > it really does not matter. Also, the concentrations seem to > be an overkill. Long ago, I soaked some 3 MM in just > TE + 1% SDS, dried and spotted 5 ul of several overnight > cultures. A year after, storing the paper in the drawer, all > clones gave lots of transformants after electroporation. > > This is a good candidate for inclusion into lab generics Wiki > found athttp://methodsandreagents.pbwiki.com/ > > DK From defilipp from ohsu.edu Fri Jul 18 17:04:05 2008 From: defilipp from ohsu.edu (defilipp@ohsu.edu) Date: Fri Jul 18 17:47:28 2008 Subject: Retroviral vectors?? Message-ID: Hi, I?m looking for retroviral or lentiviral vectors that express GFP or luciferase downstream of a multiple cloning site into which I can insert a promoter or my choice. I need both luciferase and GFP but on separate plasmids. I?m sure these must exist but am having trouble locating some. Any help would be much appreciated!! defilipp@ohsu.edu From tk from mit.edu Fri Jul 18 21:36:36 2008 From: tk from mit.edu (Tom Knight) Date: Sat Jul 19 07:25:03 2008 Subject: Protocol for identification of unknown bacteria References: Message-ID: "Justin Baiardo" writes: > Mr. Coutts- > My name is Justin Baiardo and I teach microbiology at a high school out here > in New Mexico. > I read your post on bio.net regarding a protocol for bacterial > identification through 16S PCR and then electrophoresis to compare banding > patterns of 'unknowns' that i will supply the kids to knowns that we have in > the lab. > 1. I would like a protocol for how to do this. I have heard that the haeIII > restriction enz is a good one to use. > 2. I dont know how to get 16s primers. I've done searches on the internet > and I cant seem to find them. I have heard that the BSF-343 and BMB-C are > good ones to use. > 3. I dont need to sequences, just PCR, restriction digest, then run the > gels. Here's a list of references and primers for 16S sequencing: http://openwetware.org/wiki/Bacterial_species_identification From janshar258 from yahoo.com Mon Jul 21 13:55:31 2008 From: janshar258 from yahoo.com (Sharon Rho) Date: Tue Jul 22 19:21:28 2008 Subject: Yellow EDTA ph8, 0.5M Message-ID: <884779.76592.qm@web53501.mail.re2.yahoo.com> Has anyone had problems with EDTA turning a clear?yellow solution? I used the same protocol that others in the lab used: 186.1 g of EDTA, add 500 ml of distilled water, then add NaOH pellets until a pH of 8 is reached...but it still turns out yellow. Does anyone know how to solve the problem? Thanks. ? From hroychow from nmsu.edu Tue Jul 22 21:54:36 2008 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Wed Jul 23 09:39:57 2008 Subject: Yellow EDTA ph8, 0.5M In-Reply-To: <884779.76592.qm@web53501.mail.re2.yahoo.com> References: <884779.76592.qm@web53501.mail.re2.yahoo.com> Message-ID: <2021.71.210.238.48.1216781676.squirrel@webmail.nmsu.edu> It is common, and varies from batch to batch. Usually it is not a problem. If using for sensitive rxns, simply filter it through a 0.2micron filter. > Has anyone had problems with EDTA turning a clear yellow solution? I used > the same protocol that others in the lab used: 186.1 g of EDTA, add 500 ml > of distilled water, then add NaOH pellets until a pH of 8 is reached...but > it still turns out yellow. Does anyone know how to solve the problem? > Thanks. > > > > > _______________________________________________ > 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 ivanoov from gmail.com Wed Jul 23 02:59:33 2008 From: ivanoov from gmail.com (chovek69) Date: Wed Jul 23 09:40:04 2008 Subject: Yellow EDTA ph8, 0.5M References: Message-ID: On Jul 21, 9:55?pm, Sharon Rho wrote: > Has anyone had problems with EDTA turning a clear?yellow solution? I used the same protocol that others in the lab used: 186.1 g of EDTA, add 500 ml of distilled water, then add NaOH pellets until a pH of 8 is reached...but it still turns out yellow. Does anyone know how to solve the problem? Thanks. > ? I suppose the EDTA powder is old...Is it clearly white or is yellowish ? From nobody from nospam.not Wed Jul 23 05:59:48 2008 From: nobody from nospam.not (Han) Date: Wed Jul 23 09:40:36 2008 Subject: Yellow EDTA ph8, 0.5M References: Message-ID: chovek69 wrote in news:dfae68a1-9aa7-4648-8578-f1f5e555295e@b1g2000hsg.googlegroups.com: > On Jul 21, 9:55 pm, Sharon Rho wrote: >> Has anyone had problems with EDTA turning a clear yellow solution? I >> us > ed the same protocol that others in the lab used: 186.1 g of EDTA, add > 500 ml of distilled water, then add NaOH pellets until a pH of 8 is > reached...but it still turns out yellow. Does anyone know how to solve > the problem? Thanks. >>   > > I suppose the EDTA powder is old...Is it clearly white or is > yellowish ? > Or there is tea in the system, or the NaOH pellets were really old ... -- Best regards Han email address is invalid From R.Jayakumar from roswellpark.org Wed Jul 23 08:22:42 2008 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Wed Jul 23 09:40:42 2008 Subject: Yellow EDTA ph8, 0.5M In-Reply-To: <884779.76592.qm@web53501.mail.re2.yahoo.com> References: <884779.76592.qm@web53501.mail.re2.yahoo.com> Message-ID: <97101976F8A044468CA74FE11883B90E173E7AD2@VISTA.roswellpark.org> Which company did you buy it from?? Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Sharon Rho Sent: Monday, July 21, 2008 2:56 PM To: methods@magpie.bio.indiana.edu Subject: Yellow EDTA ph8, 0.5M Has anyone had problems with EDTA turning a clear?yellow solution? I used the same protocol that others in the lab used: 186.1 g of EDTA, add 500 ml of distilled water, then add NaOH pellets until a pH of 8 is reached...but it still turns out yellow. Does anyone know how to solve the problem? Thanks. ? _______________________________________________ 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 ffog from upo.es Thu Jul 24 08:19:45 2008 From: ffog from upo.es (FILIPPO FOGLIA) Date: Thu Jul 24 20:35:47 2008 Subject: dna strider Message-ID: <3b0c073678b.48888f81@upo.es> hi, i'm a phd student in developmental biology. i'm looking for the vesion 1.4 of dna strider, i've got it in spain,but i'm in UK for a short stage and i cannot install properly the program in my mac,here. so i cannot work.. in spain i installed 1.1 and then i changed the application file 1.4f6 but now i cannot get yhis file and when people from my lab in spain sent me, it do not work. do you know a way in chich i can get a good version? thanks Filippo Foglia Centro Andaluz de Biolog?a del Desarrollo, CSIC/Universidad Pablo de Olavide, Carretera de Utrera, Km 1, 41013 Sevilla Spain http://www.upo.es/CABD/ 34 954977914 (office) 34 954348686 (Lab) Fax 34 954349376 From dprieto2006 from gmail.com Thu Jul 24 13:45:28 2008 From: dprieto2006 from gmail.com (dprieto2006@gmail.com) Date: Thu Jul 24 20:35:53 2008 Subject: DNA crosslinking on nylon memberane References: <2387671c0807161339j146896e1q62cfce5df96d9946@mail.gmail.com> <6ec6lkF618tlU1@mid.individual.net> Message-ID: <7d63509b-8820-4c9f-aa4f-159de3e30a01@z6g2000pre.googlegroups.com> On 18 jul, 15:45, Kyle Legate wrote: > Tracy Chow wrote: > > Hi Rory, > > > Thanks for your suggestion...really appreciate. > > > May I ask if you use Hybond-N+ or Hybond-XL (also positively charged) > > membrane? ?May I ask for the conditions you dry the membrane (room > > temperature ?or vacuum)? ?How long do you *dry the membranes *prior to > > hybridization? ?How long do you *pre-hybridize* prior to probe incubation? > > > THANKS!!! > > > Tracy > > I'm not Rory, but I'll toss in my 0.5 cents anyway. Both membranes will > be fine, but you crosslink them differently. N+ is nitrocellulose, XL is > nylon, so heat or UV, respectively. Dry the membrane on some filter > paper or other uncontaminated holding device by RT evaporation until > there's no visible moistness before proceeding. The time it takes to > clean up the blotting set-up should be enough time. If protected in a > plastic sleeve or a book they can be stored dry for months before > hybridizing, or they can be used immediately after crosslinking (~10 > minutes after obtaining them). I used Church buffer for a pre-hyb > solution and 1 hour in a hybridization oven (65C) was sufficient. Enough > time to label your probe. Hi, you can also fix your nucleic acids by means o MW radiation using a common microwave oven. See this paper: Nucleic Acids Res. 23:879-880, 1995 hope this can help, Daniel (dprieto@fcien.edu.uy) From t.harrison from ucl.ac.uk Fri Jul 25 05:59:10 2008 From: t.harrison from ucl.ac.uk (t.harrison@ucl.ac.uk) Date: Fri Jul 25 12:05:56 2008 Subject: hsp70 qPCR Message-ID: <7c61ca7b-0b54-4f33-81aa-9b3eef89b082@w1g2000prk.googlegroups.com> Does anyone have a method for Taqman PCR quantification of human hsp70? Thanks, Tim From gerchman from research.haifa.ac.il Fri Jul 25 14:02:04 2008 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Fri Jul 25 16:21:18 2008 Subject: dna strider (FILIPPO FOGLIA) Message-ID: <1217012524.488a232c277d4@webmail.haifa.ac.il> Write an e-mail to the author: cmarck at cea.fr Last time I checked it was a $200 software, NOT a freeware. Still $200 is pretty cheap for professional MolBio SW. For free alternatives google "CLC free" Also try http://mekentosj.com/programs/ Many other out there. Yoram G. ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From novalidaddress from nurfuerspam.de Sun Jul 27 07:46:28 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Sun Jul 27 13:55:01 2008 Subject: statistical question on sequencing project Message-ID: <942089cd-b88a-415f-8851-9c8b75cc75fc@y38g2000hsy.googlegroups.com> Dear Experts, stupid scientist wants to quantify some gene expression in his samples with pyrosequencing. The technology he plans to use offers him the possibility to identify a certain number (N) of transcripts per sample (at least 10.000). He has some ideas on how many different transcripts there might be (about 100) in one sample. Now, he is looking for a method that tells him how to calculate the number of sequences he has to obtain per sample in order to identify transcripts of which the expression is different from sample to sample. Example: If there is a transcript present at 1% in one sample, how many transcripts does he need to identify and count in order to detect a change in its relative expression of e.g. 100% (i.e. presence of 2% or 0.5% in another sample with a certain probability (say p<0.05)? Looking forward to your suggestions (textbook, paper, weblink, how this statistical problem is named etc.) Wo From ucgatan from ucl.ac.uk Mon Jul 28 05:17:29 2008 From: ucgatan from ucl.ac.uk (Tom Anderson) Date: Mon Jul 28 12:45:38 2008 Subject: statistical question on sequencing project In-Reply-To: <942089cd-b88a-415f-8851-9c8b75cc75fc@y38g2000hsy.googlegroups.com> References: <942089cd-b88a-415f-8851-9c8b75cc75fc@y38g2000hsy.googlegroups.com> Message-ID: On Sun, 27 Jul 2008, WS wrote: > Dear Experts, That's not me, but i'll contribute my P = 0.02 anyway ... > stupid scientist wants to quantify some gene expression in his samples > with pyrosequencing. The technology he plans to use offers him the > possibility to identify a certain number (N) of transcripts per sample > (at least 10.000). > > He has some ideas on how many different transcripts there might be > (about 100) in one sample. Now, he is looking for a method that tells > him how to calculate the number of sequences he has to obtain per sample > in order to identify transcripts of which the expression is different > from sample to sample. > > Example: If there is a transcript present at 1% in one sample, how many > transcripts does he need to identify and count in order to detect a > change in its relative expression of e.g. 100% (i.e. presence of 2% or > 0.5% in another sample with a certain probability (say p<0.05)? > > Looking forward to your suggestions (textbook, paper, weblink, how this > statistical problem is named etc.) I *think* this is a matter of what's called 'statistical power': http://en.wikipedia.org/wiki/Statistical_power However, this is a fairly complicated subject, and i can't really tell you any more than that. You could try the sci.math.stat newsgroup, or asking your librarian. I suspect you might need to know something about the error characteristics of the counting method, which will mean running each sample multiple times and calculating mean and standard deviation or similar. 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 editor from gene-quantification.info Mon Jul 28 09:48:57 2008 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Mon Jul 28 13:48:35 2008 Subject: qPCR Newsletter July 2008 - main focus on qPCR optimisation Message-ID: qPCR Newsletter July 2008 - main focus on qPCR optimisation Dear researcher, dear Gene Quantification page reader, Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene Quantification homepage. The focus of this newsletter issue is: - Update - Directory page - Steps and variables of a successful mRNA quantification using real- time RT-PCR - How to optimize your qPCR - qPCR Event calendar 2008: meetings & application workshops ---------------------------------------------------------------------------= ----- If this newsletter is not displayed correctly by your email client, please use following link: http://qPCRnews.gene-quantification.info/ ---------------------------------------------------------------------------= ----- Steps and variables of a successful mRNA quantification using real- time RT-PCR http://www.gene-quantification.de/optimization2.html ---------------------------------------------------------------------------= ----- How to optimize your quantitative real-time PCR PROTOCOL - Current problems in quantitative real-time RT-PCR. by T. Nolan, RE. Hands & SA Bustin; Nature Protocols (2006) Vol. 1, No. 3; p1559-1582 http://www.gene-quantification.de/nolan-hands-bustin-2006.pdf The real-time reverse transcription polymerase chain reaction (RT- qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a =91=91gold=92=92 standard, it is far fr= om being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. The widespread use of this technology has resulted in the development of numerous protocols that generate quantitative data using: fresh, frozen or archival FFPE (formalin-fixed, paraffinembedded) samples, whole-tissue biopsies, microdissected samples, single cells, tissue culture cells, total or mRNA, - a range of different cDNA priming strategies, - different enzymes or enzyme combinations, - assays of variable efficiency, sensitivity and robustness, - diverse detection chemistries, reaction conditions, thermal cyclers and - individual analysis and reporting methods. This obvious lack of standardization at every step of the assay (Figure 1) is exacerbated by significant differences in sample processing, use of controls, normalization methods and quality control management and has serious implications for the reliability, relevance and reproducibility of RT-qPCR. An overview of the considerations relating to procedures and alternative steps for carrying out the RT- qPCR reaction is shown in Figure 2. ---------------------------------------------------------------------------= ----- New papers qPCR optimisation - Evaluation of probe chemistries and platforms to improve the detection limit of real-time PCR. - Diagnostic PCR: validation and sample preparation are two sides of the same coin. - Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability. - Real-time RT-PCR: considerations for efficient and sensitive assay design. - How to reduce primer dimers (3 papers) - Standardization and quality control of PCR analyses. - Standardization of real-time PCR gene expression data from independent biological replicates. - Control of Contamination Associated with PCR and Other Amplification Reactions. - Enhancing the efficiency of a PCR using gold nanoparticles. - Increasing Detection of Polymerase Chain Reaction (PCR) by Isolation of PCR Products (IPCRp). - Increased efficiency of genetic profiling through quantity and quality assessment of fluorescently labeled oligonucleotide primers. - Primers with 5' flaps improve real-time PCR. - A real-time polymerase chain reaction-based evaluation of cDNA synthesis priming methods. - Comparison of quantitative competitive polymerase chain reaction=96 enzyme-linked immunosorbent assay with LightCycler-based polymerase chain reaction for measuring cytomegalovirus DNA in patients after hematopoietic stem cell transplantation. - Performance evaluation of thermal cyclers for PCR in a rapid cycling condition. - Sensitivity comparison of real-time PCR probe designs on a model DNA plasmid. - Real-time RT-PCR and SYBR Green I melting curve analysis for the identification of Plum pox virus strains C, EA, and W: Effect of amplicon size, melt rate, and dye translocation. - Comparison of two standardisation methods in real-time quantitative RT-PCR to follow Staphylococcus aureus genes expression during in vitro growth. ---------------------------------------------------------------------------= ----- 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 SYMPOSIUM BENELUX The prominent and still growing place taken by real-time quantitative PCR in applied and fundamental research and clinical diagnostics almost appears obvious. However, it is clear that contributions made by various scientists and companies in the field during the last decade rendered this technology useful and affordable for many users. More info =3D> http://www.gene-quantification.de/meetings.html#benelux Importantly, the qPCR domain is still in constant evolution, making it sometimes hard to stay informed about new methodological approaches or original studies using the real-time PCR. Therefore, we have scheduled a one day "Benelux qPCR Symposium" on October 6th 2008, giving the opportunity to the scientific community to get informed and discuss various aspects of real-time PCR (including but not limited to new applications, assay optimization and validation, new technologies, etc.). Scientific talks, posters sessions and industrial booths will be at the menu. Download poster =3D> http://www.gene-quantification.de/qpcr_benelux_poster= .pdf ---------------------------------------------------------------------------= ----- qPCR Symposium USA 10. - 13. November 2008 Clarion Hotel San Francisco Airport, Millbrae, CA , USA More info =3D> http://www.gene-quantification.de/meetings.html#qpcr_usa - High throughput platforms: High throughput applications, real-time RT-PCR arrays, digital PCR - Forthcoming technologies: Immuno PCR, Methylation sensitive PCR, SNP analysis, High resolution melt, microRNA detection, - Multiplex technologies - Single-cell qPCR: Pre-amplification techniques, sub-cellular PCR, Expression heterogeneity, laser microdissection, FACS sorting, Enrichment of rare cells - Multimarker diagnostics: Disease markers, Tissue specific markers, Cancer markers, Stem cells, Differentiation markers, Cancer stem cells - Real-time PCR Expression Profiling: multivariate and multiway expression profiling, temporal expression profiling, spatiotemporal maps - Pre-analytical Steps: Sampling technologies, Extraction methods, Reverse Transcription, Quality Control, Standards, Standard Operating Procedures, Interlaboratory Exercises - Normalization & Standardization: Normalization strategies, Reference genes, Spikes, Standard curves, multiplexing, inter-run calibrators, quantification strategies, mRNA degradation - Data management and data treatment: software applications, data mining, data visualization, biostatistics, multivariate statistics ---------------------------------------------------------------------------= ----- qPCR WORKSHOP TATAA Biocenter Germany - qPCR Application workshops At the TATAA Biocenter Germany we offer qPCR application workshops, the 3-day Core Module and a 2-day Biostatistics Module. qPCR courses are held in regularly in G=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 courses are held in cooperation with the Institute of Physiology, located at the Technical University of Munich, in Freising-Weihenstephan, near Munich, very close to the Munich Airport (MUC). For more information and to register for the qPCR application workshops, please see our web page: http://tataa.gene-quantification.info/ Course Occasions summer and autumn 2008: 25-29 Aug Prague qPCR Core Module + Practical Biostatistics 8-12 Sep G=F6teborg Sample Preparation + qPCR Core Module 15 - 19 Sep Freising Germany qPCR Core Module + Biostatisticsy (English language ) 13-17 Oct Prague RNA Isolation + qPCR Core Module + HRM 13-17 Oct Freising Germany qPCR Core Module + Biostatistics (Kurs wird in DEUTSCH gehalten, German language) 27-31 Oct G=F6teborg qPCR Core Module + HRM + Biostatistics 17-21 Nov Prague qPCR Core Module + Practical Biostatistics 24-28 Nov Freising Germany qPCR Core Module + Biostatistics (English language) 1-5 Dec G=F6teborg qPCR Core Module + Biostatistics 15-19 Dec Prague RNA Isolation + Expression Profiling and Data Analysis Download list of TATAA courses in autumn and fall 2008 Please register here =3D> http://www.tataa.com/Courses/Courses.html ---------------------------------------------------------------------------= ----- Forward Please send the qPCR NEWS to further scientists and friends who are interested in qPCR ! Best regards, Michael W. Pfaffl responsible Editor of the Gene Quantification Pages http://www.gene-quantification.info ---------------------------------------------------------------------------= ----- If this newsletter is not displayed correctly by your email client, please use following link: http://qPCRnews.gene-quantification.info/ The qPCR NEWS and the Gene Quantification Pages are educational sites with the only purpose of facilitating access to qPCR related information on the internet. The qPCR NEWS and the Gene Quantification Pages are edited by Michael W. Pfaffl and powered by BioScience Events. Copyright =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 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 may from genomatix.de Wed Jul 30 04:33:59 2008 From: may from genomatix.de (Klaus May) Date: Wed Jul 30 12:44:12 2008 Subject: statistical question on sequencing project References: <942089cd-b88a-415f-8851-9c8b75cc75fc@y38g2000hsy.googlegroups.com> Message-ID: <3eecfdfc-ce1f-4416-a549-6e59198a83fd@l42g2000hsc.googlegroups.com> On Jul 27, 2:46 pm, WS wrote: > Dear Experts, > > stupid scientist wants to quantify some gene expression in his samples > with pyrosequencing. The technology he plans to use offers him the > possibility to identify a certain number (N) of transcripts per sample > (at least 10.000). > > He has some ideas on how many different transcripts there might be > (about 100) in one sample. Now, he is looking for a method that tells > him how to calculate the number of sequences he has to obtain per > sample in order to identify transcripts of which the expression is > different from sample to sample. > > Example: If there is a transcript present at 1% in one sample, how > many transcripts does he need to identify and count in order to detect > a change in its relative expression of e.g. 100% (i.e. presence of 2% > or 0.5% in another sample with a certain probability (say p<0.05)? > > Looking forward to your suggestions (textbook, paper, weblink, how > this statistical problem is named etc.) > Hi! Have a look at this science paper, especially the supplementary material http://www.sciencemag.org/cgi/content/abstract/1160342 There are some cosiderations about sequencing depth. Cheers From L.Kamalian from liverpool.ac.uk Wed Jul 30 10:44:23 2008 From: L.Kamalian from liverpool.ac.uk (Kamalian, Laleh) Date: Wed Jul 30 12:44:22 2008 Subject: Invasion assay in suspension cells Message-ID: <31DC2DFFD70C174B8E3072803F894F91046CC0@EVSSTAFF1.livad.liv.ac.uk> Hi. Has anyone ever performed invasion assay in cells which grow in suspension. I would appreciate your help. L. From may from genomatix.de Wed Jul 30 04:41:39 2008 From: may from genomatix.de (Klaus May) Date: Wed Jul 30 12:45:33 2008 Subject: Analysis of Next Generation Sequencing data Message-ID: <2de56267-8681-4812-9e2f-6f1303d6fcb9@e53g2000hsa.googlegroups.com> Dear Group members, a recent Science publication: "A Global View of Gene Activity and Alternative Splicing by Deep Sequencing of the Human Transcriptome" http://www.sciencemag.org/cgi/content/abstract/1160342 demonstrates capabilities of two products from Genomatix www.genomatix.de Genomatix delivers powerful turnkey solutions downstream of Next Generation Sequencing (NGS) devices, following the Genomatix tradition of highest scientific standards and cutting edge technology. Genomatix solutions work with data from all available NGS system providers: Illumina=B4s Genome Analyzer (Solexa), Roche (454), The SOLiD TM System by Applied Biosystems, and HeliScope by Helicos. Genomatix Mapping Station (GMS) delivers ultra-fast mapping of sequence reads of any leng