From sissi_2009 from hotmail.com Mon Mar 2 13:14:20 2009 From: sissi_2009 from hotmail.com (Sissi zhang) Date: Mon Mar 2 17:28:08 2009 Subject: shaking incubator Message-ID: Dear members: Has anyone used the following shaking incubator for E. coli culture? How do you like it? Dose it work to your satisfaction? I am looking for information on this shaking incubator. Any comments would be greatly appreciated. Thanks=2C Sissi Lab Companion=AE Digital Incubating/Refrigerating Shakers=2C 83L BENCHTOP H= EATED SHAKING INCUBATOR - 120VAC / 60Hz _________________________________________________________________ Windows Live=99 Contacts: Organize your contact list.=20 http://windowslive.com/connect/post/marcusatmicrosoft.spaces.live.com-Blog-= cns!503D1D86EBB2B53C!2285.entry?ocid=3DTXT_TAGLM_WL_UGC_Contacts_032009= From novalidaddress from nurfuerspam.de Tue Mar 3 04:04:45 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Tue Mar 3 13:57:54 2009 Subject: Sodium Sulfate Precipitation Problem Message-ID: <871086e0-36fb-44d0-9d11-7f29f17fa49d@j39g2000yqn.googlegroups.com> Dear Colleagues, I am about to precipitate some purified IgG by dialysis against 18% m/ v sodium sulfate (as I want to label the protein with an amino reactive reagent, I cannot use ammonium sulfate, it's a convenient quencher). I get a huge amount of precipitate after incubating at room temp over night. However, the precipitate is floating on top of the solution, especially after centrifugation (2000g for 20 min at 25 degC). Any idea how I could obtain an ordinary pellet where one would expect it? Many thanks and best regards, Wolfgang From rdiazg from ipb.csic.es Tue Mar 3 07:10:09 2009 From: rdiazg from ipb.csic.es (=?ISO-8859-1?Q?Rosario_D=EDaz?=-=?ISO-8859-1?Q?Gonz=E1lez?=) Date: Tue Mar 3 17:18:56 2009 Subject: Genomic library construction troubleshooting Message-ID: <20090303120232.M75385@ipb.csic.es> Hi everybody, I'm working on a genomic library construction for 2-h, but I'm unable to get any insert in my vector. I'm working with pACT2 vector, digested with Bam HI and dephosphorylated with CIAP (also tried with SAP). The insert is a partially Sau3A digested genomic DNA, and ligated with different ratios (1:1, 1:3, 1:5). Both the vector and the insert are agarose-cleaned by Qiaex, and the ligase is EtOH ppt.... but all the few colonies I got are empty, w/o any insert in. Anything I'm missing? Any tip or hint? Thanks a lot in advance. Rose From R.Jayakumar from roswellpark.org Tue Mar 3 17:37:04 2009 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Tue Mar 3 19:02:09 2009 Subject: Genomic library construction troubleshooting In-Reply-To: <20090303120232.M75385@ipb.csic.es> References: <20090303120232.M75385@ipb.csic.es> Message-ID: <97101976F8A044468CA74FE11883B90E173E7C55@VISTA.roswellpark.org> Why is the ligase ethanol precipitated? Cant you buy pure ligase from NEB or from some company. And secondly, try not to use CIAP. It can cause more problems than it resolves if it is not completely deactivated. Assuming that after the ligation, the BAMHI sites are lost on the new vector+insert, redigest the ligated mix with BamHI, which should cut only the self ligated constructs (along with a small percentage of Sau3AI inserts which had BamHI sites), and then use the ligated mix for transformation. You will be selecting for more transformed clones in this case. It will be a good idea to do large scale colony hybridization screening with your probe of interest to select for clones that have your inserts of interest than doing individual plasmid preps. Saves time in screening the library as well. Wouldn't it be a better idea to use lambda systems from Stratagene which can take larger insert sizes, screen the plaques for your insert of interest and then subclone them into plasmid vectors. Think about it. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Rosario D?az-Gonz?lez Sent: Tuesday, March 03, 2009 7:10 AM To: methods@magpie.bio.indiana.edu Subject: Genomic library construction troubleshooting Hi everybody, I'm working on a genomic library construction for 2-h, but I'm unable to get any insert in my vector. I'm working with pACT2 vector, digested with Bam HI and dephosphorylated with CIAP (also tried with SAP). The insert is a partially Sau3A digested genomic DNA, and ligated with different ratios (1:1, 1:3, 1:5). Both the vector and the insert are agarose-cleaned by Qiaex, and the ligase is EtOH ppt.... but all the few colonies I got are empty, w/o any insert in. Anything I'm missing? Any tip or hint? Thanks a lot in advance. Rose _______________________________________________ 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 shifalich from rediffmail.com Wed Mar 4 04:31:00 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Wed Mar 4 12:04:10 2009 Subject: Genomic library construction troubleshooting Message-ID: <20090304093100.59080.qmail@f4mail-234-117.rediffmail.com> ?Can you consider trying TA cloning using Promega's pGEMT easy kit? I have used this for cloning my cDNA library. Promega claims cloning of fragments as big as 10Kb or so. On Tue, 03 Mar 2009 Rosario D?az-Gonz?lez wrote : >Hi everybody, > >I'm working on a genomic library construction for 2-h, but I'm unable to get >any insert in my vector. > >I'm working with pACT2 vector, digested with Bam HI and dephosphorylated with >CIAP (also tried with SAP). The insert is a partially Sau3A digested genomic >DNA, and ligated with different ratios (1:1, 1:3, 1:5). Both the vector and >the insert are agarose-cleaned by Qiaex, and the ligase is EtOH ppt.... but >all the few colonies I got are empty, w/o any insert in. > >Anything I'm missing? Any tip or hint? > >Thanks a lot in advance. > >Rose > >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From shifalich from rediffmail.com Wed Mar 4 04:36:01 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Wed Mar 4 12:04:20 2009 Subject: Sodium Sulfate Precipitation Problem Message-ID: <20090304093601.23592.qmail@f4mail-234-238.rediffmail.com> ? Try pptn. with ammonium sulphate ( abt. 33% for IgG) I have done that! It gives good results. Add slowly, keeping constantly stiriing on ice. Then keep stirring in cold room overnight and the ppt.( NIce pellet)after 10K spin at 4C for 30 minutes, can then be dissolved in water and dialized against whatever buffer you want! Hope it helps! Shifali On Tue, 03 Mar 2009 WS wrote : >Dear Colleagues, > >I am about to precipitate some purified IgG by dialysis against 18% m/ >v sodium sulfate (as I want to label the protein with an amino >reactive reagent, I cannot use ammonium sulfate, it's a convenient >quencher). I get a huge amount of precipitate after incubating at room >temp over night. However, the precipitate is floating on top of the >solution, especially after centrifugation (2000g for 20 min at 25 >degC). >Any idea how I could obtain an ordinary pellet where one would expect >it? > >Many thanks and best regards, > >Wolfgang >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From chemdude321 from gmail.com Wed Mar 4 15:14:25 2009 From: chemdude321 from gmail.com (Josh Levin) Date: Wed Mar 4 18:11:50 2009 Subject: Sodium Sulfate Precipitation Problem References: <871086e0-36fb-44d0-9d11-7f29f17fa49d@j39g2000yqn.googlegroups.com> Message-ID: <5af6c0e3-c3db-4399-bfb8-4dcabe3bc035@r36g2000vbp.googlegroups.com> On Mar 3, 4:04?am, WS wrote: > Dear Colleagues, > > I am about to precipitate some purified IgG by dialysis against 18% m/ > v sodium sulfate (as I want to label the protein with an amino > reactive reagent, I cannot use ammonium sulfate, it's a convenient > quencher). I get a huge amount of precipitate after incubating at room > temp over night. However, the precipitate is floating on top of the > solution, especially after centrifugation (2000g for 20 min at 25 > degC). > Any idea how I could obtain an ordinary pellet where one would expect > it? > > Many thanks and best regards, > > Wolfgang Hi Wolfgang-- is it necessary to precipitate the IgG? You might try concentrating it in a Centricon or some other miniconcentrator. In my experience the IgG will denature after precipitating. Typically concentration prior to amino conjugation works pretty well. Josh Levin ResearchDesignExperts From engelbert_buxbaum from hotmail.com Wed Mar 4 17:21:49 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Mar 4 18:11:55 2009 Subject: 2D gel or MS? for comparison proteomic References: Message-ID: Am 25.02.2009, 12:29 Uhr, schrieb Zhong Silin : > I want to compare total protein level from two types of plant cells. > Since my lab is made up of geneticists, I dont think we can do this > experiment on our own. I would prefer to find a company who provide such > services or collaborate with some other proteomic groups. > > Before contacting them, could some one give me some idea about this type > of experiment? I only know people have used 2D gel and MS/MS to compare > protein levels in arabidopsis roots. It is not either/or, but one after the other. First you separate the proteins, then you analyse by MS/MS. The classic method for separation is 2D-electrophoresis, but multi-dimensional chromatography (look for "MuDPIT") has also been described. From engelbert_buxbaum from hotmail.com Wed Mar 4 17:27:21 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Mar 4 18:12:00 2009 Subject: 2D gel or MS? for comparison proteomic References: Message-ID: Am 26.02.2009, 08:56 Uhr, schrieb shifali chatrath : > we do all kinds of proteomic techniques. Also, a lot of mol. Bio. stuff. > and as far as I know, 2D and MS/MS can only be used you define the > composition of proteome in two types of cells (If you meant so....) but > not to quantitate. Oh yes, 2D-electrophoresis can be used to compare levels of proteins in different samples, this is called "differential gel electrophoresis" (DIGE). From each sample you take an aliquote and label it with fluorescent dyes (or stable isotopes). An additional dye is used to label a mixture of all samples (internal standard). All samples are then mixed and run on the same gel. Then the fluorescence of a spot at different wavelengths is measured, the ratio is proportional to the ratio of proteins in the samples. From shifalich from rediffmail.com Thu Mar 5 00:22:48 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Thu Mar 5 12:19:32 2009 Subject: 2D gel or MS? for comparison proteomic Message-ID: <20090305052248.702.qmail@f4mail214.rediffmail.com> ? Yeah! I later checked with my colleagues.He also told me the same thing DIGE. But he also told its very cumbersome! Shifali On Thu, 05 Mar 2009 Dr Engelbert Buxbaum wrote : >Am 26.02.2009, 08:56 Uhr, schrieb shifali chatrath : > > >>we do all kinds of proteomic techniques. Also, a lot of mol. Bio. stuff. and as far as I know, 2D and MS/MS can only be used you define the composition of proteome in two types of cells (If you meant so....) but not to quantitate. > >Oh yes, 2D-electrophoresis can be used to compare levels of proteins in different samples, this is called "differential gel electrophoresis" (DIGE). From each sample you take an aliquote and label it with fluorescent dyes (or stable isotopes). An additional dye is used to label a mixture of all samples (internal standard). All samples are then mixed and run on the same gel. Then the fluorescence of a spot at different wavelengths is measured, the ratio is proportional to the ratio of proteins in the samples. >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From blackhole from abuse.plus.com Thu Mar 5 04:17:53 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Thu Mar 5 12:19:58 2009 Subject: Genomic library construction troubleshooting References: Message-ID: Historians believe that in newspost on Tue, 3 Mar 2009, Rosario D?az-Gonz?lez penned the following literary masterpiece: >Anything I'm missing? Any tip or hint? What is the genomic and what is the E.coli. You are using an mcr, mrr and hsdR minus E.coli? See appendix of NEB catalogue, just in case you are starting from highly methylated DNA. I take it you have tested the following. Cut vector with BamH I, no CIP, no ligase - should get back no colonies. Now ligate and should get back thousands of colonies. All should have intact BamH I site. Just proves that BamH I and ligase are working as they should etc. Insert should self ligate - test by agarose gel - proves Sau3A I generates ligatable ends. Doesn't prove they are still GATC but it's step in the right direction. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From rdiazg from ipb.csic.es Thu Mar 5 13:38:10 2009 From: rdiazg from ipb.csic.es (=?ISO-8859-1?Q?Rosario_D=EDaz?=-=?ISO-8859-1?Q?Gonz=E1lez?=) Date: Thu Mar 5 15:16:42 2009 Subject: Genomic library construction troubleshooting In-Reply-To: <20090304093100.59080.qmail@f4mail-234-117.rediffmail.com> References: <20090304093100.59080.qmail@f4mail-234-117.rediffmail.com> Message-ID: <20090305183159.M50159@ipb.csic.es> Thanks all for the tips. Unfortunately, I can't not ride off of CIAP since my IP is really fond of with that enzyme, don't tell him about trying SAP. The same happens with the EtOH ppt, he told me to do it that way. The genome is from a protozoan, and the E. coli strain we're using for the cloning is DH5alpha. The thing is that dephosphorylation works fine, it doesn't religate again but the few colonies i get are w/o any kind of insert, in fact it doesn't seem even to self-ligate: i even tried to pre-warm the vector-insert mix @ 68?, 3 min, to prevent the spontaneous self-ligation, but no better result. The control reaction (dephosphorylated vector + ligase) plate is empty, and the ligation plates have about 20 colonies... I'm thinking it might be the insert,but don't get what neither why! About the TA cloning, I considered it but how i got it into pGEM, with A-coiling? And wouldn't it be more difficult to get into the double-hybrid peyd vector with two different enzymes -those I need to use to get back the insert- when with one enzyme I'm going crazy? Maybe I can try, since I would jump over the CIAP :-) Thanks again to everybody, guys. On 4 Mar 2009 09:31:00 -0000, shifali ?chatrath wrote > ? Can you consider trying TA cloning using Promega's pGEMT easy kit? I have used this for cloning my cDNA library. Promega claims cloning of fragments as big as 10Kb or so. > > On Tue, 03 Mar 2009 Rosario D?az-Gonz?lez wrote : > >Hi everybody, > > > >I'm working on a genomic library construction for 2-h, but I'm unable to get > >any insert in my vector. > > > >I'm working with pACT2 vector, digested with Bam HI and dephosphorylated with > >CIAP (also tried with SAP). The insert is a partially Sau3A digested genomic > >DNA, and ligated with different ratios (1:1, 1:3, 1:5). Both the vector and > >the insert are agarose-cleaned by Qiaex, and the ligase is EtOH ppt.... but > >all the few colonies I got are empty, w/o any insert in. > > > >Anything I'm missing? Any tip or hint? > > > >Thanks a lot in advance. > > > >Rose > > From haydee.lopez from gmail.com Fri Mar 6 01:16:20 2009 From: haydee.lopez from gmail.com (Haydee Lopez) Date: Fri Mar 6 12:01:51 2009 Subject: Plasmid Digestion Problem Message-ID: Dear all, After I extract my plasmid I usually run a sample to be sure the plasmid it= s fine and once there I=92m sure that my plasmid its fine I continue with the next step and here comes my problem; I tried to digest the vector pPICZalphaA + my insert with different enzymes and I obtain only a smeared band. I use as a control the same vector without the insert and works perfectly, the buffer, enzyme even the extraction method are the same. I incubate both plasmids with only the buffer without enzyme and the result was the same. I think that probably was contamination with DNases but only one plasmid has been affected, so I=92m not so sure. I tried with others mi= ni preps and nothing changes. After the extraction the plasmid looks great in agarose gel. The strain there I=92m using is TOP10. If anyone has an idea o= f what=92s happening I really appreciate your help. Thank you and best regards. HL From editor from gene-quantification.info Fri Mar 6 10:41:38 2009 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Fri Mar 6 12:01:58 2009 Subject: qPCR Event Newsletter - March 2009 Message-ID: <99cee393-bc3e-4d43-8a5b-98c14490cba7@r29g2000vbp.googlegroups.com> qPCR Event Newsletter - March 2009 ----------------------------------------------- Dear researcher, dear Gene Quantification page reader, We want to inform you about the upcoming qPCR events, workshops and symposia, in spring & summer 2009. -------------------------------------------------------------------------------- TATAA Biocenter Germany - qPCR Application workshops At the TATAA Biocenter Germany we offer qPCR application workshops, the 3-day Core Module and a 2-day Biostatistics Module. qPCR courses are held in regularly in G?teborg, Sweden, in English and in Freising- Weihenstephan, Germany, in German and English, and in Prague, Czech Republic in English and Czech. Depending on the occasion the workshop language and the different prices may apply. Further customized workshops and specialized trainings will be held as well across Europe and world-wide. TATAA Biocenter Germany courses are held in cooperation with the Institute of Physiology, located at the Technical University of Munich, in Freising-Weihenstephan, near Munich, very close to the Munich Airport (MUC). For more information and to register for the qPCR application workshops, please see our web page: http://tataa.gene-quantification.info/ In April we offer two new course modules: microRNA & single-cell qPCR New courses are held in cooperation with Qiagen (microRNA) and Advalytix/Olympus (single-cell qPCR). http://www.gene-quantification.de/newsletter-March-April-2009.pdf Course Occasions spring & summer 2009: 20 - 22 April 2009 (English) NEW microRNA qPCR (Mon- Wed) 27 - 29 April 2009 (English) NEW singel-cell qPCR Application Workshop (Mon- Wed) 11 - 15 May 2009 (Deutsch) 3-day qPCR Core Module (Mon-Wed) & 2-day BioStatistics Module (Thu- Fri) 15 - 19 June 2009 (English) 3-day qPCR Core Module (Mon- Wed) & 2- day BioStatistics Module (Thu- Fri) 13 - 15 July 2009 (English) NEW microRNA qPCR (Mon- Wed) 27 - 31 July 2009 (English) 3-day qPCR Core Module (Mon- Wed) & 2- day BioStatistics Module (Thu- Fri) Here you find a lot more international courses from TATAA Biocenter / Please register here => http://www.tataa.com/Courses/Courses.html Forward Please send the qPCR NEWS to further scientists and friends who are interested in qPCR ! Best regards, Michael W. Pfaffl Manager TATAA Biocenter Germany http://TATAA.gene-quantification.info -------------------------------------------------------------------------------- Liebe Kollegen, F?r den real-time PCR Workshops vom 11. - 15. Mai 2009 am TATAA Biocenter Germany sind noch einzelne Pl?tze frei! Wir bieten ein 3-t?giges qPCR Basis Modul f?r Anf?nger und fortgeschrittene real-time PCR User an, sowie ein 2-t?giges Biostatistik und Expression Profiling Modul f?r fortgeschrittene real- time PCR User an. Sie arbeiten an verschiedenen real-time Ger?ten (Eppendorf realplex, Bio-Rad iQ5, Roche Lightcycler 2.0, und Corbett Research RotorGene 6000) in den modernen Laboren des TATAA Biocenter Germany am Start-Up Center der F?rdergesellschaft IZB GmbH, in Freising am Campus der TU- M?nchen Weihenstephan. Agenda 3-t?giges qPCR Basis Modul Inhalte qPCR Basis Modul: qPCR Theorie und Praxis, Primer und Sonden Design, Optimierung von qPCR Kontrollen, Quantifizierungsstrategien (absolute- und relative- Quantifizierung), Effizienzkorrektur, RNA und DNA Extraktion, Prinzip der RT, verschiedene Priming Methoden, SNP, Detektion, immuno-qPCR, etc... http://www.gene-quantification.de/Tagesablauf_Core_Module_3_FS.pdf Agenda 2-t?giges Biostatistik und Expression Profiling Modul Inhalte Biostatistik und Expression Profiling Modul: Einf?hrung und ?berblick in die Statistik, grundlegende und fortgeschrittene statistische Verfahren, lineare Regression, ANOVA, multiples Testen, statistische Auswertung von qPCR Datens?tzen, Expression Profiling, PCA, Self Organising Maps, Neural Networks, etc... http://www.gene-quantification.de/Tagesablauf_Biostat_Module_2_FS.pdf Im April bieten wir Ihnen zwei neue qPCR Kursmodule an: microRNA & single-celll qPCR Die Kurse weden in Kooperation mit Qiagen (microRNA) oder Advalytix/ Olympus (single-cell qPCR) gehalten. http://www.gene-quantification.de/newsletter-March-April-2009.pdf Bitte registrieren Sie sich unter der folgenden Link => http://www.bioEPS.com Im Weiteren bieten wir Englischsprachige Kurse weltweit an: Liste weiterer internationaler Workshops im Fr?hjahr & Sommer 2009 http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pdf wir freuen uns auf ein Wiedersehen in Freising, mit feundlichen Gr?ssen, Michael W. Pfaffl Manager TATAA Biocenter Germany http://TATAA.gene-quantification.info -------------------------------------------------------------------------------- The qPCR NEWS and the Gene Quantification Pages are educational sites with the only purpose of facilitating access to qPCR related information on the internet. The qPCR NEWS and the Gene Quantification Pages are edited by Michael W. Pfaffl. Copyright ? 2005 - 2009 All rights reserved. Any unauthorized use, reproduction, or transfer of this message or its contents, in any medium, is strictly prohibited. Disclaimer & Copyrights are displayed on the homepage www.gene-quantification.com To subscribe or change your e- mail address in qPCR NEWS, and if you would like to receive future issues FREE of charge, please send an e-mail with the subject SUBSCRIBE to mailto:newsletter@gene-quantification.info? subject=SUBSCRIBE From virashkgupta from gmail.com Sun Mar 8 05:59:50 2009 From: virashkgupta from gmail.com (Virash Gupta) Date: Sun Mar 8 13:32:04 2009 Subject: Methods Digest, Vol 46, Issue 6 In-Reply-To: <200903071704.n27H4BG29675@net.bio.net> References: <200903071704.n27H4BG29675@net.bio.net> Message-ID: inactivate CIAP in the presence of 2.5mM EDTA at 65C then clean with Qiaex. On 3/7/09, methods-request@oat.bio.indiana.edu < methods-request@oat.bio.indiana.edu> wrote: > > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. Re: Genomic library construction troubleshooting (DK) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Fri, 06 Mar 2009 22:23:37 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: Genomic library construction troubleshooting > To: methods@net.bio.net > Message-ID: > > In article , > "=?ISO-8859-1?Q?Rosario_D=EDaz?=-=?ISO-8859-1?Q?Gonz=E1lez?=" > wrote: > >Thanks all for the tips. Unfortunately, I can't not ride off of CIAP since > my > > IP is really fond of with that enzyme, don't tell him about trying SAP. > > Does he want to just stick to his principles or get the job done? > Leave out CIAP. In 90% cases it creates more problems than it > solves. > > DK > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 46, Issue 6 > ************************************** > -- 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 Sun Mar 8 09:07:43 2009 From: virashkgupta from gmail.com (Virash Gupta) Date: Sun Mar 8 13:32:12 2009 Subject: Shaking incubator Message-ID: Not a great thing to think about so much. Any shaker providing controlled temperature with minimum 120 rpm (~180 rpm) preferably with tube holders in the inclined position can be good enough for actively growing E.coli cultures. On 3/3/09, methods-request@oat.bio.indiana.edu < 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. shaking incubator (Sissi zhang) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Mon, 2 Mar 2009 10:14:20 -0800 > From: Sissi zhang > Subject: shaking incubator > To: Biosci Bionet news group > Message-ID: > Content-Type: text/plain; charset=3D"Windows-1252" > > > Dear members: > > > > Has anyone used the following shaking incubator for E. coli culture? > How do you like it? Dose it work to your satisfaction? I am looking for > information on this shaking incubator. Any comments would be greatly > appreciated. Thanks, Sissi > > > > Lab Companion=AE Digital Incubating/Refrigerating Shakers, 83L BENCHTOP > HEATED SHAKING INCUBATOR - 120VAC / 60Hz > _________________________________________________________________ > Windows Live=99 Contacts: Organize your contact list. > > http://windowslive.com/connect/post/marcusatmicrosoft.spaces.live.com-Blo= g-cns!503D1D86EBB2B53C!2285.entry?ocid=3DTXT_TAGLM_WL_UGC_Contacts_032009 > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 46, Issue 2 > ************************************** > --=20 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 corti.federico2 from gmail.com Tue Mar 10 11:09:00 2009 From: corti.federico2 from gmail.com (Federico Corti) Date: Tue Mar 10 12:53:09 2009 Subject: Dopamine Measurment in PC12 Message-ID: <3473fd6d0903100909o53b36c64mea7d81af62d5607b@mail.gmail.com> Dear members, I am measuring Dopamine release from NGF-differentiated PC12. I am using HPLC with C18 reverse phase column in 10 % methanol isocratic elution. It seem that after 3-4 times the column get clotted, the pressure go high and we need to wash a lot with metanol (much more than regular wash). Sometimes the injection needle is blocked and it takes time to clean it. Is anyone knows a better way to wash the column after meausiring dopamine from PC12 supernatant? In my lab. some people measure norepinephrine from synaptosomes and after measurment HPLC works well without problems. Thanks Fede From novalidaddress from nurfuerspam.de Tue Mar 10 13:08:59 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Tue Mar 10 14:12:13 2009 Subject: Dopamine Measurment in PC12 References: Message-ID: <9dbf8a79-693b-4b56-b7dd-9d3ced4451bf@j38g2000yqa.googlegroups.com> Hi Federico, seems to me you get a precipitate that blocks the system. you could a) filter your sample through 0.2?M syringe filters b) bring your sample to 10% MeOH and spin any precipitating material down before injecting (better before step a) if you consider b) c) use SPE columns for sample cleanup. Should work with methods for catecholamine isolation from blood. You might refer to the clinical chemistry department of your favorite hospital d) use a small column of the same type as pre-column to protect the actual HPLC column e) reverse your column every 2nd or so run. Best regards, Wo From RLDennis from gmail.com Tue Mar 10 15:25:59 2009 From: RLDennis from gmail.com (chicken_brain) Date: Tue Mar 10 16:05:45 2009 Subject: hplc problem Message-ID: <69cce409-5e4b-4e76-81a5-a988646c0c07@e2g2000vbe.googlegroups.com> Has anyone ever seen an HPLC output sine waves before? I've been working with the same ESA Coulochem system for about 5 years and this is a new one on me. Just running pumps and cells one day it started producing sine waves. I figured it might be a mechanical interference, I shut off all other things nearby except the machine itself and a nearby freezer, didn't help. I shut down the entire system (pumps, cells, software, everything), brought it back up and it was normal again. A little while later when analyzing samples sine waves were produced after the mobile phase spikes in a blank, and a few standards (the peaks for the standards were also evident within the noise. This sine wave noise had fairly high and consistent peaks over time (with 8 channels, channel 8 had the largest peaks, 1 had the smallest) but frequency was consistent and similar to what I saw when running just pumps without samples. Eventually it went away and the rest of the samples ran perfectly. Please let me know if anyone has ever seen this before and how you fixed it. Any brainstorms would be welcome too! From novalidaddress from nurfuerspam.de Tue Mar 10 16:25:19 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Tue Mar 10 18:30:42 2009 Subject: hplc problem References: <69cce409-5e4b-4e76-81a5-a988646c0c07@e2g2000vbe.googlegroups.com> Message-ID: <41a48706-edbf-428f-b42b-aae8922ba301@b16g2000yqb.googlegroups.com> > channel 8 had the largest peaks, 1 had the smallest Hm. A bad / loose cable, shielding, grounding comes into my mind? Interference from a machine like realtime PCR, mass spec, warp generator etc (with high periodic currents / magnetic fields) in the lab on the other side of the wall or connected to the same circuit in your building? Trapped airbubbles? A worn out piston or pulsation damper? If the frequency of your sine waves is related to the flow rate, then the problem rather might be in your machine, if not rather somewhere in outer space :) Just some thoughts. Wo From emschaub from gmail.com Tue Mar 10 16:49:33 2009 From: emschaub from gmail.com (emschaub) Date: Tue Mar 10 18:30:47 2009 Subject: Firmware for MJ Tetrad.. Help! I bricked it! Message-ID: So, I'm new, but am frantically trying to find a solution since I bricked our MJ Research Tetrad! I was at Biorad website and saw there was a firmware upgrade, and figured that I'd be a good customer and upgrade our machine just in case there were some bugs that we haven't found. Typically this is a good policy for just about all computer equipment I've ever owned... until now. So, I download it and run the upgrade utility on a computer via the serial cable. The utility goes through 95% of the way, formats, starts to install, and then makes it all the way to the end before it tells me that our hardware is too old and incompatible with this firmware upgrade. No warning, nothing in the instructions, nothing on the website that we had obsolete equipment for the firmware until this. So now it's a major paper weight. Anyone have any idea? I already called Biorad and they are saying it's our fault and want $3,000 for a service call (not including equipment replacement) and say they need to replace the motherboard. When I turn on the machine it just says that it's missing software and needs service. So, I think all we need is the old firmware and a method of getting it on the machine and things would be fine. Anyone have any ideas or any leads on anything that might be helpful? I wish I could find someone who actually wrote the firmware. I haven't been very happy with Biorad--technical support is lacking. We received a somewhat mean voicemail from them yesterday. Eric From novalidaddress from nurfuerspam.de Wed Mar 11 05:42:59 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Mar 11 12:31:44 2009 Subject: Firmware for MJ Tetrad.. Help! I bricked it! References: Message-ID: Hi Eric, first, if still possible, don't panic (too much). Has the update utility by chance created a backup of the old firmware? Or is it somewhere on a disk with it that came with the instrument? If yes, you may restore that file hopefully quite easily. Maybe you even can access a helpful engineer from Biorad or the company they bought the machine from who can dig out the file you need from the archives. If not, you need to find someone who is familiar with computer electronics and someone who own a model as close as possible to yours. Depending on the situation with the hardware, there might several options to transplant the firmware from an identical machine, eg by reading and flashing the EEPROM on an external device. Maybe it's possible to access the machine's BIOS with sort of terminal program and issue commands to dump a the firmware from a healthy machine on a disk an use this for flashing yours. HTH a bit Wolfgang From R.Jayakumar from roswellpark.org Wed Mar 11 14:05:47 2009 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Wed Mar 11 14:54:19 2009 Subject: Firmware for MJ Tetrad.. Help! I bricked it! In-Reply-To: References: Message-ID: <97101976F8A044468CA74FE11883B90E173E7C61@VISTA.roswellpark.org> I don't like the Bio-Rad people at all. Their equipments are lousy and so is their technical support. I nowadays buy all our stuff from other companies like Bio-Tek (great equipments) and others. What better way to make money than to put up a vague firmware support and missle people into installing it and then charge money for repairing the damage. I am sure that it just cleared the BIOS. A good computer tech should be able to do it. Or you can just get a run down MJ research cycler (I doubt it, since MJ Research people made some of the best PCR machines I have ever used), and scavenge it for the BIOS chip. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of emschaub Sent: Tuesday, March 10, 2009 5:50 PM To: methods@magpie.bio.indiana.edu Subject: Firmware for MJ Tetrad.. Help! I bricked it! So, I'm new, but am frantically trying to find a solution since I bricked our MJ Research Tetrad! I was at Biorad website and saw there was a firmware upgrade, and figured that I'd be a good customer and upgrade our machine just in case there were some bugs that we haven't found. Typically this is a good policy for just about all computer equipment I've ever owned... until now. So, I download it and run the upgrade utility on a computer via the serial cable. The utility goes through 95% of the way, formats, starts to install, and then makes it all the way to the end before it tells me that our hardware is too old and incompatible with this firmware upgrade. No warning, nothing in the instructions, nothing on the website that we had obsolete equipment for the firmware until this. So now it's a major paper weight. Anyone have any idea? I already called Biorad and they are saying it's our fault and want $3,000 for a service call (not including equipment replacement) and say they need to replace the motherboard. When I turn on the machine it just says that it's missing software and needs service. So, I think all we need is the old firmware and a method of getting it on the machine and things would be fine. Anyone have any ideas or any leads on anything that might be helpful? I wish I could find someone who actually wrote the firmware. I haven't been very happy with Biorad--technical support is lacking. We received a somewhat mean voicemail from them yesterday. Eric _______________________________________________ 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 mrnance from lsi.umich.edu Fri Mar 13 07:40:50 2009 From: mrnance from lsi.umich.edu (Mark Nance) Date: Fri Mar 13 10:37:34 2009 Subject: Firmware for MJ Tetrad Message-ID: <49BA1C12020000B500012E84@lsigroupwise01.lsi.umich.edu> Bio-Rad's mail-in service is much less expensive than their on-site service. At this point, you should talk to Bio-Rad through your sales rep instead of tech support. Tech support has no incentive to help, but your sales rep wants your future business. I don't know that they will give you the old software for free, but they might let you mail in your tetrad so that they can put the old software on. Explain specifically that you think the hardware is fine and that it is most likely a software problem; Bio-Rad sells enough products that the phone operators don't understand how all of them work. Re-explaining it as a software re-install problem that could be done at their leisure in California could be much cheaper than paying for an airplane ticket and 24 hours of tech time for a service call. NEVER do a firmware upgrade unless there is a very good reason for it. Many software upgrades make sense, and can be undone, but firmware upgrades are an opportunity for you to brick your computer and are rarely reversible. Good luck, Mark From lsbquest from yahoo.co.uk Sun Mar 15 07:02:23 2009 From: lsbquest from yahoo.co.uk (Lori Buetow) Date: Sun Mar 15 14:24:52 2009 Subject: Problems with purifying and concentrating a GST-fusion protein Message-ID: <682980.24388.qm@web24705.mail.ird.yahoo.com> Hi, I am currently trying to purify a protein from a bacterial expression of a GST-fusion construct of the protein. When my protein is eluted from a glutathione column, it co-elutes with a band of approximately 60 kDa in size -- I think the co-eluted protein is the chaperone GroEL. My protein is a homolog of another protein with 97% sequence identity that has been expressed and purified from the same vector/bacterial system and the authors identified their co-eluted protein as GroEL. When my GST-fusion protein is eluted from a glutathione column, it has a 260/280 ratio of approximately 1. Fractions with high concentrations of protein have high absorbance readings at 320nm but there is no visible evidence of precipitate in solution. In addition, when I concentrate the GST-fusion protein, the 320 increases as though the protein is precipitating, but there is no visible evidence of precipitate in solution. After cleaving the GST tag while dialyzing, the absorbance readings are comparable to pre-cleavage, suggesting that small molecules are not contributing factors to the strange absorbance readings. Is this characteristic of a soluble aggregate or is a characteristic of a chaperone/protein interaction? I have tried the prep 3 times. The first two times, I was able to obtain purified protein but the yields were too low for sufficient experimental work. The third time I tried the prep I had better yields initially (GST-fusion protein) but concentration of the protein was very slow and dramatically decreased the yield. For the third prep, I had optimized expression levels (about a 10-fold improvement over the 1st two) and I had used more pellet. Has anyone seen this type of behavior with a GST-fusion protein before and does anyone have any advice on how to increase the soluble/functioning yields from this prep? Thanks, Laurie From fatemeh_2554 from yahoo.com Mon Mar 16 05:14:03 2009 From: fatemeh_2554 from yahoo.com (Fatemeh Azadegan) Date: Mon Mar 16 12:04:54 2009 Subject: (no subject) Message-ID: <942664.72607.qm@web59515.mail.ac4.yahoo.com> hello,can anyone tell me how to extract dna fram blood (human)stored in a ordinary freezer.i have to separate blood mixed by serum.anyone have a protocol for it with soulting out method.the samples are six month old. From ivanoov from gmail.com Fri Mar 20 05:09:50 2009 From: ivanoov from gmail.com (chovek69) Date: Fri Mar 20 11:34:46 2009 Subject: (no subject) References: Message-ID: <4f5a63a8-8d1a-4143-b148-bff27d664c2e@v38g2000yqb.googlegroups.com> On Mar 16, 12:14?pm, Fatemeh Azadegan wrote: > hello,can anyone tell me how to extract dna fram blood (human)stored in a ordinary freezer.i have to separate blood mixed by serum.anyone have a protocol for it with soulting out method.the samples are six month old. You can not separate serum because freezed-thawed blood would be haemolyzed. However you can isolate DNA from the whole thing. See this thread http://www.protocol-online.org/biology-forums/posts/5198.html From ngeraci from gmail.com Fri Mar 20 15:27:12 2009 From: ngeraci from gmail.com (Nicholas Geraci) Date: Fri Mar 20 15:48:30 2009 Subject: Human skin DNA extraction protocol Message-ID: <975ec7520903201327y42a42108hb8cb169770e7b5e9@mail.gmail.com> I'm looking for a reliable protocol to extract gDNA from human skin biopsies. We plan on doing methylation analysis on samples from this tissue type. Would anyone have a protocol they would be willing to share or a reference to which you could point me? -- Nicholas S. Geraci, MS Research Associate Children's Memorial Research Center, Chicago From sudhee26 from gmail.com Sun Mar 22 09:33:50 2009 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Sun Mar 22 12:07:32 2009 Subject: Human skin DNA extraction protocol In-Reply-To: <975ec7520903201327y42a42108hb8cb169770e7b5e9@mail.gmail.com> References: <975ec7520903201327y42a42108hb8cb169770e7b5e9@mail.gmail.com> Message-ID: Hi, http://jmd.amjpathol.org/cgi/content/full/3/1/22would this be of any help to you? Sudheendra. On Sat, Mar 21, 2009 at 1:57 AM, Nicholas Geraci wrote: > I'm looking for a reliable protocol to extract gDNA from human skin > biopsies. We plan on doing methylation analysis on samples from this > tissue > type. Would anyone have a protocol they would be willing to share or a > reference to which you could point me? > > -- > Nicholas S. Geraci, MS > Research Associate > Children's Memorial Research Center, Chicago > _______________________________________________ > 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 ntravers from medicina.unige.it Wed Mar 25 09:59:19 2009 From: ntravers from medicina.unige.it (Nicola Traverso) Date: Wed Mar 25 11:45:16 2009 Subject: native PAGE pH Message-ID: <49CA46C7.70005@medicina.unige.it> Dear Dr Engelbert Buxbaum I found this e-adress in bio.net. I'm a little bit confused about pH in PAGE gels, in particular native gels. I need your help. When in SDS-PAGE (discontinuous system) I use -tris-glycine pH 8.3 as electrophoretic buffer -tris-HCl pH 6.8 as stacking buffer -tris-HCl pH 8.8 as resolving buffer at which pH are proteins separated? I though at 8.8, i.e. the pH of the resolving buffer, but I've recently read that they separate at pH 9.5! How can this be true? Does the pH of the tris-glycine electrophoretic buffer influence the pH of the resolving gel during the electrophoretic run? if yes, how much? In native-PAGE (continuous system) -do I have to use the same pH in sample buffer, electrophoretic buffer and resolving buffer? Tris-HCl is OK? Must I use tris-glycine in the electrophoretic buffer? May I change the pH, let us say, between 7 and 10 as I like? In native-PAGE (discontinuous system) -how can I set systems at pH different than usual (stacking: 6.8; resolving 8.8; electrophoretic 8.3)? for exemple, if I desire to resolve at pH 7.8, how do I have to prepare the other buffers (stacking, sample, and electrophoresis)? Thank you in advance Best regards Nicola Traverso Dept Experimental Medicine Section of General Pathology University of Genova Italy From gerchman from research.haifa.ac.il Wed Mar 25 14:23:54 2009 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Wed Mar 25 15:58:12 2009 Subject: Universal anti-aquaporine antibody? Message-ID: <1238009034.49ca84ca909c8@webmail.haifa.ac.il> Hello all We are trying to follow aquaporin expression in arthropods. Does anyone have an idea for a anti aquaporin antibody (commercial or otherwise) that will react with multiple aquaporins? Many thanks Yoram gerchman at research dot haifa dot ac dot il ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From editor from gene-quantification.info Thu Mar 26 05:29:10 2009 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Thu Mar 26 10:46:15 2009 Subject: qPCR NEWS March 2009 - focus on HRM & HRM dyes Message-ID: qPCR NEWS March 2009 - focus on HRM & HRM dyes --------------------------------------------------------------------- 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: - HRM page updated with new papers - NEW page - HRM dyes - Online translation service of the Gene Quantification - New qPCR events in autumn 2009 - New qPCR workshop modules at the TATAA Biocenter Germany - Online translation service of the Gene Quantification => http://translation.gene-quantification.info/ New qPCR workshop modules at the TATAA Biocenter Germany => http://tataa.gene-quantification.info/ European wide qPCR application workshops => register now ! => course program spring - summer 2009 => http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pdf -------------------------------------------------------------------------------- High Resolution Melting (HRM) is a novel, homogeneous, close-tube, post-PCR method, enabling genomic researchers to analyze genetic variations (SNPs, mutations, methylations) in PCR amplicons. It goes beyond the power of classical melting curve analysis by allowing to study the thermal denaturation of a double-stranded DNA in much more detail and with much higher information yield than ever before. HRM characterizes nucleic acid samples based on their disassociation (melting) behavior. Samples can be discriminated according to their sequence, length, GC content or strand complementarity. Even single base changes such as SNPs (single nucleotide polymorphisms) can be readily identified. http://hrm.gene-quantification.info/ The most important High Resolution Melting application is gene scanning - the search for the presence of unknown variations in PCR amplicons prior to or as an alternative to sequencing. Mutations in PCR products are detectable by High Resolution Melting because they change the shape of DNA melting curves. A combination of new- generation DNA dyes, high-end instrumentation and sophisticated analysis software allows to detect these changes and to derive information about the underlying sequence constellation. HRM Applications => new papers The introduction of HRM has renewed interest in the utility of DNA melting for a wide range of uses, including: Mutation discovery (gene scanning) Screening for loss of heterozygosity DNA fingerprinting SNP genotyping Characterization of haplotype blocks DNA methylation analysis DNA mapping Species identification Somatic acquired mutation ratios HLA compatibility typing Association (case/control) studies Allelic prevalence in a population Identification of candidate predisposition genes http://hrm.gene-quantification.info/ -------------------------------------------------------------------------------- High Resolution Melting Dyes & probes http://hrm-dyes.gene-quantification.info/ The secret of High Resolution Meling (HRM) analysis is to monitor this process happening in real-time. This is achieved by using saturating fluorescent dye. HRM dyes are known as intercalating dyes bind specifically and in high amount (= saturating dye, except SYBR Green) to double-stranded DNA and when they are bound they fluoresce brightly. In the absence of double-stranded DNA they have nothing to bind to and they only fluoresce at a low level (estimations are between 1-4% false positive fluorescence). At the beginning of the HRM analysis, after complete PCR amplification, there is a high level of fluorescence in the sample because of the millions of copies of the amplicon. But as the sample DNA is heated up and the two strands of the DNA denaturate there is no longer any double stranded DNA present and thus fluorescence is reduced => http://hrm-dyes.gene-quantification.info/ * LC Green * SYTO9 * Eva Green * BEBO * SYBR Green -------------------------------------------------------------------------------- online translation Since October 2008 we provide an online translation service of the Gene Quantification pages to several languages. Please recognize this is an automatic and robotic based translation service, and therefore we can give NO guarantee about the generated content. It should help to understand the "rough" content of the Gene Quantification pages, but still the original is the ENGLISH version: http://translation.gene-quantification.info/ -------------------------------------------------------------------------------- With the new qPCR INFO PORTAL and all the presented tools we will help you with to find the right information about qPCR and related topics in Molecular Biology in the literature and in the World Wide Web. => Papers / Protocols / Methods / Databases / Alets / Feeds / Books / Forums / E-mail / Directory http://infoportal.gene-quantification.info/ -------------------------------------------------------------------------------- Upcoming Events World-wide academic and commercial qPCR Events http://events.gene-quantification.info/ Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars, qPCR Education Program, etc. Please submit your qPCR event here => events@gene- quantification.info -------------------------------------------------------------------------------- qPCR WORKSHOP BioEPS GmbH / TATAA Biocenter Germany - qPCR Application workshops At the TATAA Biocenter Germany we offer qPCR application workshops, a 3-day qPCR Core Module and a 2-day qPCR Biostatistics Module. All courses are held regularly in G?teborg, Sweden, in English and in Freising-Weihenstephan, Germany, in German and English, and in Prague, Czech Republic in English and Czech. Depending on the occasion the workshop language and the different prices may apply. Further customized workshops and specialized trainings will be held as well across Europe and world-wide. TATAA Biocenter Germany workshops are held in cooperation with BioEPS GmbH, located at the campus of the Technical University of Munich, in Freising-Weihenstephan, very close to the Munich Airport (MUC). For more information and registration, please see our web page: => http://TATAA.gene-quantification.info/ Course Occasions 2009: 3-day qPCR Core Module (Mon. - Wed.) 2-day BioStatistics Module (Thu. - Fri.) 3-day single-cell qPCR Module (Mon. - Wed.) 3-day microRNA Module (Mon. - Wed.) 20 - 22 April 2009 (E) NEW microRNA qPCR 11 - 15 May 2009 (Deutsch) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics Module (Thu. - Fri.) 15 - 19 Juni 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics Module (Thu. - Fri.) 13 - 15 Juli 2009 (E) NEW microRNA qPCR 27 - 31 Juli 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics Module (Thu. - Fri.) => http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pdf -------------------------------------------------------------------------------- Forward Please send the qPCR NEWS to further scientists and friends who are interested in qPCR ! Best regards, Michael W. Pfaffl responsible Editor of the Gene Quantification Pages http://www.gene-quantification.info -------------------------------------------------------------------------------- If this newsletter is not displayed correctly by your email client, please use following link: http://qpcrnews.gene-quantification.info/ The qPCR NEWS and the Gene Quantification Pages are educational sites with the only purpose of facilitating access to qPCR related information on the internet. The qPCR NEWS and the Gene Quantification Pages are edited by Michael W. Pfaffl. Copyright ? 2005 - 2009 All rights reserved. Any unauthorized use, reproduction, or transfer of this message or its contents, in any medium, is strictly prohibited. Disclaimer & Copyrights are displayed on the homepage www.gene-quantification.com To subscribe or change your e-mail address in qPCR NEWS, and if you would like to receive future issues FREE of charge, please send an e- mail with the subject SUBSCRIBE to mailto:newsletter@gene- quantification.info?subject=SUBSCRIBE From sumanknp from gmail.com Thu Mar 26 00:56:08 2009 From: sumanknp from gmail.com (Suman) Date: Thu Mar 26 10:46:23 2009 Subject: Methods Digest, Vol 46, Issue 16 In-Reply-To: <200903251705.n2PH5Lh11194@net.bio.net> References: <200903251705.n2PH5Lh11194@net.bio.net> Message-ID: <2c1410660903252256mbdd384eo17b3abe2a125729b@mail.gmail.com> Hi frens, pleae send me a copy of this method..i think its useful for me thank you On Thu, Mar 26, 2009 at 2:05 AM, 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. native PAGE pH (Nicola Traverso) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 25 Mar 2009 15:59:19 +0100 > From: Nicola Traverso > Subject: native PAGE pH > To: methods@magpie.bio.indiana.edu > Message-ID: <49CA46C7.70005@medicina.unige.it> > Content-Type: text/plain; charset=ISO-8859-15; format=flowed > > Dear Dr Engelbert Buxbaum > > I found this e-adress in bio.net. > I'm a little bit confused about pH in PAGE gels, in particular native > gels. I need your help. > > When in SDS-PAGE (discontinuous system) I use > -tris-glycine pH 8.3 as electrophoretic buffer > -tris-HCl pH 6.8 as stacking buffer > -tris-HCl pH 8.8 as resolving buffer > at which pH are proteins separated? > I though at 8.8, i.e. the pH of the resolving buffer, but I've recently > read that they separate at pH 9.5! How can this be true? > Does the pH of the tris-glycine electrophoretic buffer influence the pH > of the resolving gel during the electrophoretic run? if yes, how much? > > In native-PAGE (continuous system) > -do I have to use the same pH in sample buffer, electrophoretic buffer > and resolving buffer? Tris-HCl is OK? Must I use tris-glycine in the > electrophoretic buffer? May I change the pH, let us say, between 7 and > 10 as I like? > > In native-PAGE (discontinuous system) > -how can I set systems at pH different than usual (stacking: 6.8; > resolving 8.8; electrophoretic 8.3)? for exemple, if I desire to resolve > at pH 7.8, how do I have to prepare the other buffers (stacking, sample, > and electrophoresis)? > > Thank you in advance > > Best regards > > Nicola Traverso > Dept Experimental Medicine > Section of General Pathology > University of Genova > Italy > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 46, Issue 16 > *************************************** > -- Suman From engelbert_buxbaum from hotmail.com Fri Mar 27 12:12:08 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri Mar 27 16:35:22 2009 Subject: native PAGE pH References: Message-ID: Am 25.03.2009, 10:59 Uhr, schrieb Nicola Traverso : > When in SDS-PAGE (discontinuous system) I use > -tris-glycine pH 8.3 as electrophoretic buffer > -tris-HCl pH 6.8 as stacking buffer > -tris-HCl pH 8.8 as resolving buffer > at which pH are proteins separated? > I though at 8.8, i.e. the pH of the resolving buffer, but I've recently > read that they separate at pH 9.5! How can this be true? The answer to this question is fairly complicated, and may be found in the following papers: @article{Orn-62, AUTHOR= {L. Ornstein}, TITLE= {Disk electrophoresis: {I}. {B}ackground and theory}, YEAR= {1962}, JOURNAL= {Ann. N. Y. Acad. Sci.}, PAGES= {321-351}, VOLUME= {121}, DOI= {10.1111/j.1749-6632.1964.tb14207.x}, LANGUAGE= {engl} } @article{Jov-73a, AUTHOR= {T.M. Jovin}, TITLE= {Multiphasic Zone Electrophoresis. {I}. {S}teady-State Moving-Boundary Systems Formed by Different Electrolyte Combinations}, JOURNAL= {Biochemistry}, VOLUME= {12}, YEAR= {1973}, PAGES= {871-879}, LANGUAGE= {engl} } @article{Jov-73b, AUTHOR= {T.M. Jovin}, TITLE= {Multiphasic Zone Electrophoresis. {II}. Design of Integrated Discontinuous Buffer Systems for Analytical and Preparative Fractionation}, JOURNAL= {Biochemistry}, VOLUME= {12}, YEAR= {1973}, PAGES= {879-890}, LANGUAGE= {engl} } @article{Jov-73c, AUTHOR= {T.M. Jovin}, TITLE= {Multiphasic Zone Electrophoresis. {III}. {F}urther Analysis and New Forms of Discontinuous Buffer Systems}, JOURNAL= {Biochemistry}, VOLUME= {12}, YEAR= {1973}, PAGES= {890-898}, LANGUAGE= {engl} } @article{Jov-73d, AUTHOR= {T.M. Jovin}, TITLE= {Multiphasic zone electrophoresis. {IV} {D}esign and analysis of discontinuous buffer systems with a digital computer}, JOURNAL= {Ann. N.Y. Acad. Sci.}, VOLUME= {209}, YEAR= {1973}, PAGES= {477-496}, LANGUAGE= {engl} } From cjmcdermottroe from googlemail.com Mon Mar 30 03:34:04 2009 From: cjmcdermottroe from googlemail.com (Chris McDermott-Roe) Date: Mon Mar 30 11:42:58 2009 Subject: protein purification & imidazole problems Message-ID: Hi all, I've expressed a recombinant protein in E.coli, purified it via Ni-NTA chromatography and am now attempting to measure its potential inhibitory activity in a fluorescence assay that involves excitation at 485 nm and measurement at 530 nm. The problem is that my protein prep is giving a huge signal (as is my buffer alone control) which makes me think the imidazole (which is present in the elution buffer) is fluorescing. Does anybody have any experience with this kind of thing? Cheers Chris From mankolkar from yahoo.co.in Mon Mar 30 05:35:20 2009 From: mankolkar from yahoo.co.in (Mandar Ankolkar) Date: Mon Mar 30 11:43:03 2009 Subject: Need help for EMSA(GElshift assay) Message-ID: <980260.6469.qm@web7605.mail.in.yahoo.com> Dear Mr. Heinz-Juergen Schaefers, ?My name is Mandar. I am a PhD student from India. I am trying to do EMSA (Gelshift assay)? for my work . However I want to bind antibody to my DNA instead of a nuclear extract or a protein. I came across your thread posted in Bionet (I know long back but still..). I was wondering can I use the same protocoland the recipies of the buffers that you have posted for my case also. I am feeling a little worried about the DTT and PMSF and triton X which will kill my antibody. Also can you tell me the significance of the MgCl2 and EDTA in the protocol. If you have any useful and article or reference on EMSA protocol, Kindly forward it to me. Waiting for your reply, Thanking You, Mandar Add more friends to your messenger and enjoy! Go to http://messenger.yahoo.com/invite/ From engelbert_buxbaum from hotmail.com Tue Mar 31 09:30:57 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Mar 31 11:55:45 2009 Subject: protein purification & imidazole problems References: Message-ID: Am 30.03.2009, 04:34 Uhr, schrieb Chris McDermott-Roe : > Hi all, > > I've expressed a recombinant protein in E.coli, purified it via Ni-NTA > chromatography and am now attempting to measure its potential inhibitory > activity in a fluorescence assay that involves excitation at 485 nm and > measurement at 530 nm. The problem is that my protein prep is giving a > huge > signal (as is my buffer alone control) which makes me think the imidazole > (which is present in the elution buffer) is fluorescing. Test the components of your buffer singly and find out which is causing the signal. Also make sure you are not looking at stray light: sample should be filtered over a 0.22 ?m low protein binding membrane filter, suitable high-pass filters in excitation and low-pass filters in emission beam. Btw, before I'd use the protein preparation I'd probably do a buffer exchange. Although His does not fluoresce, in high concentrations it will affect buffering of your samples and also have a kosmotropic (water-ordering) effect that can seriously change your proteins properties. Gel filtration, dialysis or ultrafiltration are possibilities. From sissi_2009 from hotmail.com Mon Mar 2 20:44:11 2009 From: sissi_2009 from hotmail.com (Sissi zhang) Date: Wed Sep 30 17:17:08 2009 Subject: (no subject) Message-ID: Kyle Thank you for the advice. I am still trying to figure out why my culture is not growing well. Now I just test the starter culture since it did not grow to the expected cell densities in the first place. The OD600=0.75 after 20 hr incubation at 37C at 300 rpm. The culture was half transparent after 20 hrs growth. Here was the procedure: The colony was picked from fresh agar plate. The single colony (around 1.2 mm in diameter) was inoculated to 2.5 mL LB broth (with or without Amp) After 20 hrs growth, both tubes gave me the same cell density, OD600=0.75 (with or without 100 ug/mL Amp). I checked the pH of LB broth and adjust it to pH7.5 before autoclave. I really do not know what the trouble is. Transformed DH5 +pUC19 should grow very fast. I just placed an order for liquid LB broth from different vendor (not dehydrated LB), in case the problem lies in the water I used to make LB broth or something. Could it be contamination issue? But I don't see how. The super broth formula you used seems very good. I will try it if the new LB broth I just ordered will not work. Thanks, Sissi _________________________________________________________________ Windows Live™ Contacts: Organize your contact list. http://windowslive.com/connect/post/marcusatmicrosoft.spaces.live.com-Blog-cns!503D1D86EBB2B53C!2285.entry?ocid=TXT_TAGLM_WL_UGC_Contacts_032009