From aawara from pontiff-playground.org Sun Feb 1 17:06:18 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Sun Feb 1 18:06:12 2009 Subject: Electroporation References: <6udb0pFeoar3U1@mid.individual.net> Message-ID: In <6udb0pFeoar3U1@mid.individual.net>, Christian Praetorius wrote: > dk@no.email.thankstospam.net (DK) wrote: > >>The best and easiest thing is to do nothing. 1 ul of ligation mix >>is perfectly OK with normal electroporation. > > Thats my experience, too. Making too much effort doesn't make sense. Oddly enough this has failed for me - many, many, times. We use 1 mm gap cuvettes, and 1800 volts (25 uF capacitance, and I think 200 ohms resistance). What are your settings? I would love to be able to do away with drop dialysis. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From prae from gmx.net Mon Feb 2 04:06:53 2009 From: prae from gmx.net (Christian Praetorius) Date: Mon Feb 2 12:27:04 2009 Subject: Electroporation References: <6udb0pFeoar3U1@mid.individual.net> Message-ID: <6unrddFg2336U1@mid.individual.net> Aawara Chowdhury wrote: >Oddly enough this has failed for me - many, many, times. We use 1 mm gap >cuvettes, and 1800 volts (25 uF capacitance, and I think 200 ohms >resistance). What are your settings? I used the same settings, when we did electroporation. As long as the cells were washed good enough, we never had problems with short circuits. Since we have a quite good protocol for making supercompetent cells, we moved away from it. Christian -- X-no-Sig: yes From aawara from pontiff-playground.org Mon Feb 2 06:27:21 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Mon Feb 2 12:27:09 2009 Subject: Electroporation References: <6udb0pFeoar3U1@mid.individual.net> <6unrddFg2336U1@mid.individual.net> Message-ID: In <6unrddFg2336U1@mid.individual.net>, Christian Praetorius wrote: > Aawara Chowdhury wrote: > >>Oddly enough this has failed for me - many, many, times. We use 1 mm gap >>cuvettes, and 1800 volts (25 uF capacitance, and I think 200 ohms >>resistance). What are your settings? > > I used the same settings, when we did electroporation. As long as the > cells were washed good enough, we never had problems with short > circuits. Since we have a quite good protocol for making > supercompetent cells, we moved away from it. Can you share this protocol with us? Thanks! AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From novalidaddress from nurfuerspam.de Tue Feb 3 04:11:12 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Tue Feb 3 14:29:09 2009 Subject: homemade uv254 lamp? Message-ID: <0e0748a7-87bf-43f8-b3b3-5ff4bee2d4bb@w39g2000prb.googlegroups.com> Dear Collegaues, I thought of converting a uv appliance for testing bank notes into a uv light source for examining uv254 tlc plates. Having noticed that 254nm uv tubes are used for erasing EPROMS (sort of memory chips), I consider to exchange the fluorescent tube in the moneytester. The whole setup would be less than 30 USD in contrast to a uv254 lamp from lab suppliers which is 200 USD or more. The notably difference however is that there would be no glass or filter shielding the *visible* part of the spectrum, which might result in decreased sensitivity. I would like to ask now if anyone has experience with a similar setup and might give me some advice or would like to comment. Many thanks and best regards, Wolfgang From prae from gmx.net Tue Feb 3 11:17:11 2009 From: prae from gmx.net (Christian Praetorius) Date: Tue Feb 3 14:29:14 2009 Subject: Electroporation References: <6udb0pFeoar3U1@mid.individual.net> <6unrddFg2336U1@mid.individual.net> Message-ID: <6ur906Fgq2tjU1@mid.individual.net> Aawara Chowdhury wrote: >Can you share this protocol with us? Sure: http://www3.hi.is/~pra/SuperCompetent%20bacteria_Inoue.pdf Christian -- X-no-Sig: yes From eenigoy from gmail.com Wed Feb 4 05:01:09 2009 From: eenigoy from gmail.com (yoginee budhkar) Date: Wed Feb 4 10:06:19 2009 Subject: Electroporation Message-ID: <77ddf6c70902040201j2ab13709tb813bcd1e1843960@mail.gmail.com> I have been using the same settings which are preset in the instrument. The arcing stopped after limiting the volume of ligated mix to 1/20th the volume of Electrocompetent cells. Initially I was adding 2microlit ligated mix for 20microlit comp cells, now I'm adding only 1microlit. Thanks for all the suggestions! --Yg > Message: 1 > Date: Mon, 02 Feb 2009 11:27:21 GMT > From: Aawara Chowdhury > Subject: Re: Electroporation > To: methods@net.bio.net > Message-ID: > Content-Type: text/plain; charset=us-ascii > > In <6unrddFg2336U1@mid.individual.net>, > Christian Praetorius wrote: > > > Aawara Chowdhury wrote: > > > >>Oddly enough this has failed for me - many, many, times. We use 1 mm gap > >>cuvettes, and 1800 volts (25 uF capacitance, and I think 200 ohms > >>resistance). What are your settings? > > > > I used the same settings, when we did electroporation. As long as the > > cells were washed good enough, we never had problems with short > > circuits. Since we have a quite good protocol for making > > supercompetent cells, we moved away from it. > > Can you share this protocol with us? > > Thanks! > AC > > -- > Email: echo 36434455860060025978157675027927670979097959886449930P | dc ------------------------------ > > Message: 2 > Date: Mon, 02 Feb 2009 09:06:53 +0000 > From: Christian Praetorius > Subject: Re: Electroporation > To: methods@net.bio.net > Message-ID: <6unrddFg2336U1@mid.individual.net> > Content-Type: text/plain; charset=us-ascii > > Aawara Chowdhury wrote: > > >Oddly enough this has failed for me - many, many, times. We use 1 mm gap > >cuvettes, and 1800 volts (25 uF capacitance, and I think 200 ohms > >resistance). What are your settings? > > I used the same settings, when we did electroporation. As long as the > cells were washed good enough, we never had problems with short > circuits. Since we have a quite good protocol for making > supercompetent cells, we moved away from it. > > Christian > > -- > X-no-Sig: yes > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 45, Issue 2 > ************************************** From eric.meltzer from gmail.com Wed Feb 4 05:42:03 2009 From: eric.meltzer from gmail.com (Eric Meltzer) Date: Wed Feb 4 10:06:25 2009 Subject: Fwd: Common integration loci for yeast? In-Reply-To: References: <628D3F39A80B5F47A4F231911D2B58CFCAEE8E@MSGWSDCPMB02.nyumc.org> Message-ID: Hi Jennifer, Thanks very much for the info! I hunted down some of those resources, and I think I am fairly clear on what I want to do. I figured I'd run this plan by the list in case anyone has experience with this type of screen, so I can avoid whatever pitfalls there might be. I have two constructs, both fairly small (800bp and 500bp roughly) that I want to integrate into URA3 and LYS2 loci by ends-out integration (i.e. deletion.) I want to PCR my fragments with homology tails for those two loci, then transform into URA3 and LYS2 strains. After transformation, I figure I can do 5FOA and aAA screens for the integrants. Doing both of the screens as deletion screens seems to be kind of uncommon, so I wanted to make sure I wasn't missing anything . I'm wondering whether using an 800bp construct to delete a roughly 4kbp gene is viable? It seems that at some point if your deletion construct is too small, you would be likely to get single crossover events leading to duplication over deletion. Thanks! -Eric On Wed, Jan 28, 2009 at 1:36 PM, Chang, Jennifer wrote: > > Hi, Eric, > > I hope I can be of some use here. > > When I worked in a yeast lab, we always used metabolic loci (HIS3, URA3, LEU2, TRP1, ADE2). Once we knew had a integration, it was easy to select fo= r that clone on media lacking the metabolite (screening through the different clones to find the right one is another matter). If you plan on doing something else later with these yeast cells, such as mating to a different strain or transformation with a plasmid, then you might want to consider which metabolic loci you use. Just be aware that you want your nutritional markers to be compatible at all times. From my experience, HIS is the most common one used so having the integration at a different loci might be better. > > I don't know exactly what you're using your constructs for but if you wan= t to control expression levels, perhaps integrating into the locus of an inducible (GAL1) or high-expressing gene (ADH1) is desirable? > > I can't remember the specific references but try searching for R. Rothstein. The Rothstein lab constructed many of important strains in use now and I know they must have published something on one-step or two-step gene replacement in yeast. Fred Sherman's Getting Started with Yeast (published 1993 but I'm sure there's newer versions now) was really handy when I worked in the yeast lab. > > Regards, > Jennifer Chang, PhD > Post Doctoral Fellow, Cell Biology > Medical Sciences Building, Room 698 > New York University School of Medicine > 550 First Avenue > New York, NY 10016 > Tel 212=96263-5316 > Fax 212-263-8561 > > > > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu on behalf of Eric Meltzer > Sent: Wed 1/28/2009 9:05 AM > To: methods@magpie.bio.indiana.edu > Subject: Common integration loci for yeast? > > I'm looking to integrate a series of reasonably large constructs into a > yeast strain. I know that people commonly integrate into HO, and of course > various metabolic loci like HIS or URA. In trying to figure out the best > place to integrate, I've been trying to see if there are some other commo= n > loci that don't affect growth much (or only affect growth on selective media > like HIS etc.). If anyone can point me in the direction of a good paper, or > just a few good loci, I'd appreciate it! > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > > > > ------------------------------------------------------------ > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organizatio= n accepts no liability for any damage caused by any virus transmitted by this email. > =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D=3D=3D=3D=3D=3D=3D=3D=3D > From prae from gmx.net Wed Feb 4 05:09:25 2009 From: prae from gmx.net (Christian Praetorius) Date: Wed Feb 4 10:06:51 2009 Subject: Electroporation References: <6udb0pFeoar3U1@mid.individual.net> <6unrddFg2336U1@mid.individual.net> <6ur906Fgq2tjU1@mid.individual.net> <927il.45$Vk5.16@newsfe13.iad> Message-ID: <6ut7qlFh39pcU1@mid.individual.net> dk@no.email.thankstospam.net (DK) wrote: >In my experience, transformation efficiency of Inoue cells somehow >deteriorates slowly upon storage at -70C. Unlike electroporation >"competent" cells. We usually makes batches, which are used within six month, in this time period it works. The effiency is probably going down a little bit, but this was never a problem. >We do use Inoue protocol in non-critical cases just to save on >electroporation cuvettes: take electrocompetent cells, mix with This was the main issue. >for a little longer at 37C seems to work just as well as 42C. Saves >from paranoia in the lab about someone "absolutely needing" 42C >bath NOW :-) :-) The heat shocking is something religious anyway. Ask two and they will tell you at least three different times for the heat shock. I found anything between 30sec and 2min working. >Can't resist few notes on a protocol: Thanks for it. >Gotta love people who swear by OD600. When will >biologists finally realize that OD600 is partially a function of It also depends on the photometer. >Why would any sane biologist filter sterilize pure DMSO? No. I don't do it either. >3. Snap freezing. Has anyone ever compared it to just sticking >cells at -70C? It is well-known that when freezing "real" cells I haven't compared it for cells, but its worth a try. I have to convince my colleagues only (can be hard sometimes...). >cells this way and they work well. I just wonder if the same is >applicable to Inoue cells. Probably. Things are often made more complicated than it has to be... Christian -- X-no-Sig: yes From peter.ianakiev from gmail.com Thu Feb 5 22:56:23 2009 From: peter.ianakiev from gmail.com (peter) Date: Fri Feb 6 02:28:53 2009 Subject: rolling circle amplification Message-ID: <8f131875-722c-40c7-8bbb-14a8d3713687@q30g2000prq.googlegroups.com> Dear fellow scientists, Does anyone have experience with rolling circle amplification? In particular I am interested what is the smallest circle that rolls , preferably in vivo. Thanks for the board wisdom. peter From rompubil from gmail.com Fri Feb 6 04:15:32 2009 From: rompubil from gmail.com (rompubil@gmail.com) Date: Fri Feb 6 13:42:05 2009 Subject: TNT T7 kit (Promega) with (Stratagene) SP6 pol Message-ID: Hello, My lab has the TNT T7 Coupled Reticulucyte Lysate System (Promega). However the cDNA I want to transcribe/translate is in a plasmid under the control of Sp6 promoter. My lab has the Sp6 RNA polymerase from Stratagene. Therefore, has somebody try to add to the TNT mixture a different RNA polymerase (from a different company)? I have found some posts describing a buffert that can be used in that kit. However, these posts were related to T7 RNA polymerase. I would prefer to avoid subcloning the cDNA in a different plasmid and/ or carrying out transcription/translation in two steps. Thanks Rom From peter.ianakiev from gmail.com Fri Feb 6 08:33:55 2009 From: peter.ianakiev from gmail.com (peter) Date: Fri Feb 6 13:42:12 2009 Subject: TNT T7 kit (Promega) with (Stratagene) SP6 pol References: Message-ID: <3766c7a8-089c-402a-8347-1a651306faf9@r37g2000prr.googlegroups.com> On Feb 6, 4:15?am, rompu...@gmail.com wrote: > Hello, > My lab has the TNT T7 Coupled Reticulucyte Lysate System (Promega). > However the cDNA I want to transcribe/translate is in a plasmid under > the control of Sp6 promoter. My lab has the Sp6 RNA polymerase from > Stratagene. Therefore, has somebody try to add to the TNT mixture a > different RNA polymerase (from a different company)? I have found some > posts describing a buffert that can be used in that kit. However, > these posts were related to T7 RNA polymerase. > > I would prefer to avoid subcloning the cDNA in a different plasmid and/ > or carrying out transcription/translation in two steps. > > Thanks > Rom I would not mess with the kit. Just make RNA separately with the SP6 pol , purify and use it with the reticulocyte extract. my2c From stewjw from gmail.com Fri Feb 6 11:13:05 2009 From: stewjw from gmail.com (StewJW) Date: Fri Feb 6 13:42:18 2009 Subject: DNA software question References: Message-ID: For Mac lovers out there, also check out: http://mekentosj.com/programs/ From khyate_99 from yahoo.com Fri Feb 6 15:28:24 2009 From: khyate_99 from yahoo.com (mohamed hassan) Date: Sat Feb 7 00:38:41 2009 Subject: Rhizobium Genomic DNA Extraction Message-ID: <221596.34535.qm@web54503.mail.re2.yahoo.com> Hello: could you please send me the most suitable protocol for Rhizobium genomic DNA extraction starting from cloned bacteria in a solid medium, for RAPD application. Thank you very much. khyate From b.singh121 from rediffmail.com Sat Feb 7 03:17:06 2009 From: b.singh121 from rediffmail.com (Bhupi ) Date: Sat Feb 7 16:31:03 2009 Subject: rolling circle amplification Message-ID: <1233905577.S.2852.3176.f4mail-235-205.rediffmail.com.1233994626.397@webmail.rediffmail.com> In vivo, i do not know but in -vitro i have amplified 80bp circle On Fri, 06 Feb 2009 13:02:57 +0530 wrote >Dear fellow scientists, >Does anyone have experience with rolling circle amplification? In >particular I am interested what is the smallest circle that rolls , >preferably in vivo. > >Thanks for the board wisdom. >peter >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods > From shifalich from rediffmail.com Sat Feb 7 05:41:37 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Sat Feb 7 16:35:20 2009 Subject: Bacterial growth measurement at 600nm? Message-ID: <1233689540.S.2709.59656.f4mail-235-207.rediffmail.com.old.1234003297.41790@webmail.rediffmail.com> Dear all! I got to know that Bacterial cells when suspended in PBS,their O.D. should be measured at 450 nm or so. And I used to think, we measure O.D.at 600nm because LB is yellow and we blank the plain medium at 600nm, so we measure at 600nm. On the other hand, i read in some discussion forum: When measuring light scattering it is important to consider the wavelength of light used a bacterial culture. Microorganisms may contain numerous macromolecules that will absorb light, including DNA (254 nm), proteins (280 nm), cytochromes (400-500 nm), and possible cell pigments. When measuring bacteria by light scattering it is best to pick a wavelength where absorption is at a minimum and for most bacterial cultures wavelengths around 600 nm are a good choice. However, the exact wavelength chosen is species specific. If the above statement is true then why measure at 450 or so when suspended in PBS? Any suggestions or ideas friends? Shifali On Wed, 04 Feb 2009 01:02:20 +0530 wrote >Aawara Chowdhury wrote: > >>Can you share this protocol with us? > >Sure: >http://www3.hi.is/~pra/SuperCompetent%20bacteria_Inoue.pdf > >Christian > >-- >X-no-Sig: yes >_______________________________________________ >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 legatekBLAH from hotmail.com Sun Feb 8 05:16:13 2009 From: legatekBLAH from hotmail.com (Kyle Legate) Date: Sun Feb 8 13:22:02 2009 Subject: Electroporation In-Reply-To: <6ut7qlFh39pcU1@mid.individual.net> References: <6udb0pFeoar3U1@mid.individual.net> <6unrddFg2336U1@mid.individual.net> <6ur906Fgq2tjU1@mid.individual.net> <927il.45$Vk5.16@newsfe13.iad> <6ut7qlFh39pcU1@mid.individual.net> Message-ID: <6v7pmrFiollvU1@mid.individual.net> Christian Praetorius wrote: >> for a little longer at 37C seems to work just as well as 42C. Saves >>from paranoia in the lab about someone "absolutely needing" 42C >> bath NOW :-) > > :-) > The heat shocking is something religious anyway. Ask two and they will > tell you at least three different times for the heat shock. I found > anything between 30sec and 2min working. > Heat shock is not even necessary in most cases. It depends on the strain, mostly. In my hands, SURE cells require a heat shock but DH5alpha do not. Since I use DH5alpha these days for cloning I only used heat shock for a particularly troublesome blunt ligation that was rather inefficient (heat shock does raise the transformation efficiency by about an order of magnitude, but since my competent cells are often ~10^9 I don't need it). My protocol: 1. Incubate DNA mix (ligation, supercoil, etc) with E. coli 5-10 minutes on ice. 2. Add 10X vol room temp LB and directly plate ~30 uL (for supercoil) or 100 uL (for ligation). 3. ??? 4. Profit! Kyle From gerchman from research.haifa.ac.il Sun Feb 8 15:13:48 2009 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Sun Feb 8 17:22:15 2009 Subject: Bacterial growth measurement at 600nm? Message-ID: <1234124028.498f3cfce8802@webmail.haifa.ac.il> This might help. Cyanobacteria growth is often measured at 750nm due to pigments absorbent at 600nm. Yoram ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From Gur from Fermentek.com Mon Feb 9 03:01:59 2009 From: Gur from Fermentek.com (Maksim Gur) Date: Mon Feb 9 12:56:28 2009 Subject: international units to milligram conversion Message-ID: <498FE2F7.4090301@Fermentek.com> Dear friends, perhaps this is not the best group, but How may I express 1000 international units of Thiostrepton antibiotic as milligrams of pure thiostrepton? I failed to understand the pharmacopoea From editor from gene-quantification.info Tue Feb 10 05:29:04 2009 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Tue Feb 10 09:46:06 2009 Subject: qPCR 2009 Event Agenda is online => http://online-agenda.qpcr2009.net/ Message-ID: qPCR 2009 Event Agenda is online =3D> http://online-agenda.qpcr2009.net/ The event is organized jointly by Chair of Physiology, Technische Universit=E4t M=FCnchen (TUM) and BioEPS GmbH, Freising, Germany http://www.qpcr2009.net/ The Physiology Weihenstephan at the Technische Universit=E4t M=FCnchen is organizing the 4th international qPCR 2009 Event taking place March 9 =96 13, 2009 in Freising-Weihenstephan, Germany. Scientists from all around the world will come to exchange ideas, share experiences, and discuss the exciting future of the perhaps most powerful analytical technology ever developed in the life sciences area =96 the quantitative real-time polymerase chain reaction (qPCR). 230 invited international scientists will present their latest research findings in the qPCR field. Focus of the event will be on Molecular Diagnostic and Molecular Markers. We are proud to present 230 scientific contributions (86 talks & 144 posters) at the symposium. Please find the FINAL qPCR 2009 Event Agenda, with a detailed time table where you can find the all TALK presentations: http://www.bioeps.com/qpcr2009/qpcr2009-agenda.pdf Here you find the ONLINE version of the qPCR 2009 Event Agenda, where you can find the all TALK and POSTER presentations: http://online-agenda.qp= cr2009.net/ It is a pleasure to announce the Nobel Prize Laureate Kary Mullis at the symposium in an own session =9325th Anniversary of PCR=94. qPCR is an improved form of the PCR technology that was awarded the 1993 years=92 Nobel price in Chemistry. Using qPCR the amount of target nucleic acid in a complex sample can be determined with high precision, great accuracy, excellent specificity and the ultimate sensitivity of detecting a single molecule. The technique has revolutionized all molecular sciences and diagnostic applications. Conference presentations will include high throughput applications, improved instrumentation, high performance nucleic acid extraction, immuno-qPCR applications, single-cell applications, and application involving siRNAs and microRNA. Further developments of qPCR technology that will be presented include miniaturization, high throughput platforms, cost efficacy, validity, flexibility, quality assessment and reliable data calculations and interpretation. Today there is no field in the life sciences research and diagnostics areas that has not introduced qPCR technology for nucleic acid analysis. The combination with reverse transcription enables determination of mRNA and widely opens the window for =93Transcriptomics=94 =96 the first step of gene expression and =93Functional Genomics=94. In connection with the symposium four practical qPCR Workshops will be held March 12 =96 13, 2009 by the TATAA Biocenter (www.tataa.com) - the leading qPCR service provider in Europe. The 2-day workshops are hosted by international renowned scientists and experts in the field. The workshop themes will be: (1) Classical qPCR Application Workshop; (2) qPCR Biostatistics & Expression Profiling; (3) Sample preparation & Nucleic acid extraction; (4) High-Resolution-Melt analysis & Immuno-qPCR. An Industrial Exhibition will take place parallel to the symposium, with 35 leading biotechnology companies presenting their latest developments in the PCR field, including real-time PCR cyclers, nucleic acid extraction robots, consumables, fluorescence dyes, DNA and RNA detection and amplification chemistries, as well as real-time PCR data analysis software. The Physiology Weihenstephan at the Center of Life and Food Sciences of Technische Universit=E4t M=FCnchen, chaired by Prof. Heinrich H. D. Meyer, is a leading authority in the molecular physiology of mammalian species. Cutting edge biochemical and molecular biology techniques are established for basic and applied research on the regulation of reproduction, lactation, immunology, and growth. Both traditional endocrinology and paracrine regulations are studied in numerous tissues. Dr. Michael W. Pfaffl is developing qRT-PCR methods, software algorithms and tools for quantitative gene expression analysis. He also maintains the leading qPCR information web page: http://www.gene-quantification.info For more information about the qPCR 2009 event see http://qPCR2009.net or contact Dr. Michael W. Pfaffl qPCR2009@wzw.tum.de or Dr. Martina Reiter BioEPS GmbH martina.reiter@bioeps.com From whyharish from gmail.com Wed Feb 11 14:29:15 2009 From: whyharish from gmail.com (whyharish reddy) Date: Wed Feb 11 18:11:32 2009 Subject: STABILIZERS FOR ENZYMES (mainly XYLANASE)Methods Digest, Vol 45, Issue 7 Message-ID: <5894aabe0902111129u304af63eke4f4a40b84eed718@mail.gmail.com> Hello , This is HARISH KUMAR REDDY Y, *MY PROBLEM:* *I am unable to maintain my enzyme activity not more than 2 days, * *please help in mainatining the activity of XYLANASE ENZYME for more than 2-3 months, suggest some Enzyme Stablizers?* ** *thanking you * ** *Harish Kumar Reddy Y* From GLOBAL INSTITUTE OF BIOTECHNOLOGY & OSMANIA UNIVERSITY, HYDERABAD, INDIA . ** On Mon, Feb 9, 2009 at 10:34 PM, wrote: > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. Re: Electroporation (Kyle Legate) > 2. Re: Bacterial growth measurement at 600nm? (Yoram Gerchman) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Sun, 08 Feb 2009 11:16:13 +0100 > From: Kyle Legate > Subject: Re: Electroporation > To: methods@net.bio.net > Message-ID: <6v7pmrFiollvU1@mid.individual.net> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed > > Christian Praetorius wrote: > > >> for a little longer at 37C seems to work just as well as 42C. Saves > >>from paranoia in the lab about someone "absolutely needing" 42C > >> bath NOW :-) > > > > :-) > > The heat shocking is something religious anyway. Ask two and they will > > tell you at least three different times for the heat shock. I found > > anything between 30sec and 2min working. > > > Heat shock is not even necessary in most cases. It depends on the > strain, mostly. In my hands, SURE cells require a heat shock but > DH5alpha do not. Since I use DH5alpha these days for cloning I only used > heat shock for a particularly troublesome blunt ligation that was rather > inefficient (heat shock does raise the transformation efficiency by > about an order of magnitude, but since my competent cells are often > ~10^9 I don't need it). My protocol: > > 1. Incubate DNA mix (ligation, supercoil, etc) with E. coli 5-10 minutes > on ice. > 2. Add 10X vol room temp LB and directly plate ~30 uL (for supercoil) or > 100 uL (for ligation). > 3. ??? > 4. Profit! > > Kyle > > > ------------------------------ > > Message: 2 > Date: Sun, 8 Feb 2009 22:13:48 +0200 > From: Yoram Gerchman > Subject: Re: Bacterial growth measurement at 600nm? > To: methods@oat.bio.indiana.edu > Message-ID: <1234124028.498f3cfce8802@webmail.haifa.ac.il> > Content-Type: text/plain; charset=ISO-8859-1 > > > This might help. Cyanobacteria growth is often measured at 750nm due to > pigments > absorbent at 600nm. > Yoram > > ------------------------------------------------------------------------ > This message was sent using IMP, the Webmail Program of Haifa University > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 45, Issue 7 > ************************************** > -- Y.HARISH KUMAR REDDY From ning227 from gmail.com Fri Feb 13 09:39:04 2009 From: ning227 from gmail.com (ning227@gmail.com) Date: Fri Feb 13 12:38:38 2009 Subject: storage of isopentane Message-ID: <7344bf4b-11c9-44e4-bd24-a4981e80c78d@p13g2000yqc.googlegroups.com> X-No-Archive: Yes Somone in my lab claims that isopentane (2-methylbutane) should be stored with the lid slightly unscrewed to prevent it popping open. I would have thought this is rather dangerous as surely vapours could escape and create a fire hazard, since the flash point is -51C From novalidaddress from nurfuerspam.de Fri Feb 13 13:01:43 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Fri Feb 13 16:42:06 2009 Subject: storage of isopentane References: <7344bf4b-11c9-44e4-bd24-a4981e80c78d@p13g2000yqc.googlegroups.com> Message-ID: <17125057-abcf-43e8-a57e-c390879dfaaa@l1g2000yqj.googlegroups.com> X-No-Archive: Yes Well, as isobutane boils at 28-30 degC (depending on source and atmospheric pressure :). I'd store it in a tightly sealed glass bottle which is equipped with a safety valve that prevents the buildup of dangerous overpressure which might cause the bottle to explode. Store it as cool as possible in a well ventilated place (the hood or better a cabinet for flammable liquids), but don't put it in a fridge or a cold room which is not specially constructed (i.e is EX-proof) to keep chemicals which may propose explosion hazards. Storing it in a not tigthly sealed bottle as your colleague suggests, might lead to the continuous evaporation of isopentane due to its vapor pressure and possibly the buildup of explosive mixtures with air when there is no good ventilation. Actually, in what kind of container has the manufacturer shipped it to you and isn't there any safety advice on the bottle? Best regards, Wo On 13 Feb., 15:39, ning...@gmail.com wrote: > > Somone in my lab claims that isopentane (2-methylbutane) should be > stored with the lid slightly unscrewed to prevent it popping open. > > I would have thought this is rather dangerous as surely vapours could > escape and create a fire hazard, since the flash point is -51C From engelbert_buxbaum from hotmail.com Sun Feb 15 07:39:38 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Sun Feb 15 15:13:46 2009 Subject: STABILIZERS FOR ENZYMES (mainly XYLANASE)Methods Digest, Vol 45, Issue 7 References: Message-ID: Am 11.02.2009, 15:29 Uhr, schrieb whyharish reddy : > *I am unable to maintain my enzyme activity not more than 2 days, * > *please help in mainatining the activity of XYLANASE ENZYME for more than > 2-3 months, suggest some Enzyme Stablizers?* Have you done a literature search (scholar.google.com, PubMed)? Of hand I can help only with some general hints: - Stability is temperature dependent. As is generally known, enzymes can be heat-denatured, but some are also cold-denatured. - Stability is strongly pH-dependent. Avoid storing enzymes near the pI, and at extreme pH values. Other than that, trial and error. - Some enzymes are sensitive to ionic strength and osmotic pressure. Keep both near physiological values. For intracellular enzymes use KCl, for extracellular NaCl to adjust ionic strenght. Manitol, sucrose, glycerol or similar polyols are used to maintain osmotic pressure. - Some enzymes require specific cofactors for activity and may be stabilised in their presence. Mg, Zn... - Sterile filter your preparation using a low-protein binding membrane (0.22 ?m) and use aseptic techniques for handling. Add presevatives (Na azide, Thimerosal...) but check that they are compatible with the enzyme. - Check an inactive preparation for what actually happend, for example by SDS-PAGE, light scattering and CD-spectroscopy. Is the enzyme degraded (presence of proteolytic enzymes, microorganisms), forms aggregates or changes conformation? That info can help you develop more targeted approaches to the problem. From straube from biochem.mpg.de Sun Feb 15 15:29:53 2009 From: straube from biochem.mpg.de (Werner Straube) Date: Sun Feb 15 19:41:54 2009 Subject: Detergent concentration for nuclear membrane dissolving Message-ID: <36B75E885C4376419EC0020DA151B41C01149A1B@msx.w2k.biochem.mpg.de> Hello everybody, A friend of mine is interested isolating nuclear proteins. After he has isolated the nuclei of cells he then wants to dissolve the nuclear membrane without destroying the interactions of nuclear proteins. We were already discussing the type and concentrations of detergents, however we did not get final clue. Can anyone help me out there with some suggestions or protocols or are there good references (protocols) in the literature (for general membrane dissolving and in particular nuclear membrane dissolving) ? Thank you very much, Werner From SBrown from ccia.unsw.edu.au Sun Feb 15 20:23:26 2009 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Sun Feb 15 23:56:39 2009 Subject: methylcellulose plating problem Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A02972C3D@mail01.ccia.local> Hi, This may be a long shot but i am banging my head against the wall over it. I have been doing some methocult plating, basically i take normal Nalm-6 cells, add 1.6 mls methocult, 1.2 mls FCS, 1.2mls IMDM media and then i add dexamethosone at a conc. of 8uM. Last year this would kill the majority of the Nalm-6 cells (EC50 = 6nM), there were always some colonies that would grow through so i added some methotrexate to try and decrease the formation of colonies. For the past 6 months I have been getting equal numbers of colonies in my control (the nalms) as well as my experimental population of cells. I've checked my conc. of dex, repeated MTTs to confirm the EC50, repeated the plating several times but continue to get too many colonies being formed in the control. Previous plating showed a distinct difference in the amount of colonies formed in the control and the experimental situation. Does anyone have any ideas as to what may be going on or any suggestions for an experiment i may be able to do to get to the bottom of this? cheers Scott Scott Brown PhD Candidate Molecular Carcinogenesis Program Children's Cancer Institute of Australia for Medical Research High Street (PO Box 81) RANDWICK NSW 2031 AUSTRALIA Phone: +61 2 9382 1829 Fax: +61 2 9382 1850 Email: sbrown@ccia.unsw.edu.au Web: www.ccia.org.au Children's Cancer Institute Australia is the only independent medical research institute in Australia solely devoted to research into the causes, prevention and cure of childhood cancer. Our vision is to save the lives of all children with cancer and 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. 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 Mon Feb 16 09:18:57 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Feb 16 13:05:49 2009 Subject: Detergent concentration for nuclear membrane dissolving References: Message-ID: Am 15.02.2009, 16:29 Uhr, schrieb Werner Straube : > A friend of mine is interested isolating nuclear proteins. After he has > isolated the nuclei of cells he then wants to dissolve the nuclear > membrane without destroying the interactions of nuclear proteins. In my experience positively charged detergents like CTAB can dissolve nuclei very well (better than SDS) while at the same time are generally mild (milder than OG or DM) to proteins. Whether or not the interaction that your friend is interested in is maintained under these conditions he/she will have to find out. From sagdeev from gmail.com Mon Feb 16 09:56:37 2009 From: sagdeev from gmail.com (Igor Sagdeev) Date: Mon Feb 16 13:05:55 2009 Subject: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) Message-ID: <93e7307c0902160656h4ca45af9i2c09e88303dfea2e@mail.gmail.com> Can anyone comment of the difference(s) between these products: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts)? Thank you in advance! From prae from gmx.net Tue Feb 17 04:07:47 2009 From: prae from gmx.net (Christian Praetorius) Date: Tue Feb 17 12:49:49 2009 Subject: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) References: Message-ID: <6vvd34FltmlmU1@mid.individual.net> dk@no.email.thankstospam.net (DK) wrote: >The "RNAses are so >everywhere that the only way to get rid of them is to treat everything >with some magic expensive solutions" is basically a myth. Can you tell this our technician? Christian -- X-no-Sig: yes From talulahgosh from gmail.com Tue Feb 17 08:04:29 2009 From: talulahgosh from gmail.com (talulah gosh) Date: Tue Feb 17 12:49:56 2009 Subject: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) References: <6vvd34FltmlmU1@mid.individual.net> Message-ID: Can you also tell this to our PI? :) On Feb 17, 4:07?am, Christian Praetorius wrote: > d...@no.email.thankstospam.net (DK) wrote: > >The "RNAses are so > >everywhere that the only way to get rid of them is to treat everything > >with some magic expensive solutions" is basically a myth. > > Can you tell this our technician? > > Christian > > -- > X-no-Sig: yes From shifalich from rediffmail.com Tue Feb 17 10:17:54 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Tue Feb 17 12:50:01 2009 Subject: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) Message-ID: <1234808067.S.3359.32701.f4mail-235-214.rediffmail.com.old.1234883874.64338@webmail.rediffmail.com> Both work equally well! We have used both! Shifali On Mon, 16 Feb 2009 23:44:27 +0530 wrote >Can anyone comment of the ?difference(s) between these products: > >RNase Zap (Invitrogen) > >and > > RNase Away (Molecular Bioproducts)? > >Thank you in advance! > >_______________________________________________ >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 mlsulliv from wisc.edu Tue Feb 17 13:17:59 2009 From: mlsulliv from wisc.edu (Michael Sullivan) Date: Tue Feb 17 15:50:17 2009 Subject: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) In-Reply-To: References: Message-ID: On Feb 16, 2009, at 6:16 PM, DK wrote: > In article , Igor > Sagdeev wrote: >> Can anyone comment of the difference(s) between these products: >> >> RNase Zap (Invitrogen) >> >> and >> >> RNase Away (Molecular Bioproducts)? > > Well, > > MSDS for both products makes it reasonably obvious that RNAse Away > is simply a sodium hydroxide at unspecified concentration and RNase > Zap is simply an SDS at unknown concentration. > > Take your pick. I'd contend that with already clean glassware that was > not handled by bare hands neither is necessary. The "RNAses are so > everywhere that the only way to get rid of them is to treat everything > with some magic expensive solutions" is basically a myth. > > DK > _______________________________________________ > The more one works with RNA, the one more realizes that DK's point is true! My favorite example of the extreme paranoia of RNases was a lab I knew of that baked the mortars and pestles they used to grind tissue for RNA preps! So there might be a little RNase on those: far more will be liberated when the tissue gets ground! They did go through a lot of mortars since these tended to break when cooling too fast out of the oven. As for magic solutions, I personally use dilute bleach when I feel some treatment is called for. Ambion's web site even says this is an alternative to their solution. Another hint is that usually there is no special need to treat water with DEPC. My experience is that 18 megaohm water from a milliQ system is essentially RNase free. Mike --- Michael L. Sullivan Plant Research Molecular Geneticist US Dairy Forage Research Center ARS-USDA 1925 Linden Drive West Madison, WI 53706 (608) 890-0046 (Phone) (608) 890-0076 (FAX) From hroychow from nmsu.edu Tue Feb 17 18:58:29 2009 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Tue Feb 17 20:25:43 2009 Subject: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) In-Reply-To: References: Message-ID: <1980.128.123.174.0.1234915109.squirrel@webmail.nmsu.edu> You guys have summarized the paranoia well. However, in my experience, baking the glasswares always worked rather well. Of course, whoever pours LiqN2 on the Mortar/pestle straight out of the oven has a problem far more serious than RNase in the prep. ;) Hiranya. > > On Feb 16, 2009, at 6:16 PM, DK wrote: > >> In article , Igor >> Sagdeev wrote: >>> Can anyone comment of the difference(s) between these products: >>> >>> RNase Zap (Invitrogen) >>> >>> and >>> >>> RNase Away (Molecular Bioproducts)? >> >> Well, >> >> MSDS for both products makes it reasonably obvious that RNAse Away >> is simply a sodium hydroxide at unspecified concentration and RNase >> Zap is simply an SDS at unknown concentration. >> >> Take your pick. I'd contend that with already clean glassware that was >> not handled by bare hands neither is necessary. The "RNAses are so >> everywhere that the only way to get rid of them is to treat everything >> with some magic expensive solutions" is basically a myth. >> >> DK >> _______________________________________________ >> > > The more one works with RNA, the one more realizes that DK's point is > true! > > My favorite example of the extreme paranoia of RNases was a lab I > knew of that baked the mortars and pestles they used to grind tissue > for RNA preps! So there might be a little RNase on those: far more > will be liberated when the tissue gets ground! They did go through a > lot of mortars since these tended to break when cooling too fast out > of the oven. > > As for magic solutions, I personally use dilute bleach when I feel > some treatment is called for. Ambion's web site even says this is an > alternative to their solution. > > Another hint is that usually there is no special need to treat water > with DEPC. My experience is that 18 megaohm water from a milliQ > system is essentially RNase free. > > Mike > --- > Michael L. Sullivan > Plant Research Molecular Geneticist > US Dairy Forage Research Center > ARS-USDA > 1925 Linden Drive West > Madison, WI 53706 > (608) 890-0046 (Phone) > (608) 890-0076 (FAX) > > _______________________________________________ > 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 aawara from pontiff-playground.org Tue Feb 17 21:24:03 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Wed Feb 18 10:39:31 2009 Subject: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) References: Message-ID: <7jKml.1580$Sv4.243@newsfe15.iad> In , DK wrote: > Take your pick. I'd contend that with already clean glassware that was > not handled by bare hands neither is necessary. The "RNAses are so > everywhere that the only way to get rid of them is to treat everything > with some magic expensive solutions" is basically a myth. Let me elaborate on this statement, because I take issue with it in part. My Ph.D. thesis was on the structure of retroviral RNAs. The structural analysis, obtained in part by digestion with structure/sequence specific RNases of end-labeled viral RNAs, was exquisitely sensitive to nuclease contamination. I've recovered RNase A activity in bottles that were autoclaved ..... Having said that, for 99% of uses, the background level of RNase makes no difference at all. Further, while I can appreciate that there may be a loss of signal if one does a Northern for full-length mRNA, these days, most quantitation is performed using techniques such as real-time PCR of reverse-transcribed samples. Such techniques are much less sensitive to small amounts of degradation by RNases in the environment. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From prae from gmx.net Wed Feb 18 04:28:56 2009 From: prae from gmx.net (Christian Praetorius) Date: Wed Feb 18 10:40:10 2009 Subject: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) References: Message-ID: <7022mpFmdmjbU1@mid.individual.net> Michael Sullivan wrote: >The more one works with RNA, the one more realizes that DK's point is >true! Its important to work careful and clean. But this applies to all lab work, which is done carefully. >Another hint is that usually there is no special need to treat water >with DEPC. My experience is that 18 megaohm water from a milliQ >system is essentially RNase free. Try to bring this into the minds of people. Christian -- X-no-Sig: yes From R.Jayakumar from roswellpark.org Wed Feb 18 11:55:02 2009 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Wed Feb 18 12:48:53 2009 Subject: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) In-Reply-To: References: Message-ID: <97101976F8A044468CA74FE11883B90E173E7C3D@VISTA.roswellpark.org> But RNAse contamination of the milliq water can happen when it is in transit to your reaction tubes which may also be RNAse free, and also the tubing which dispenses the water from the Milliq cartridge to your "Rnase-free" storage container. It is important the glass bottles or carboys or other plasticware that is used to store this milliQ water is clean too, wherein lies the problem that it is virtually impossible to eliminate RNAses during the transitionary phase. That is why it is always PRUDENT (an important word in the vocabulary of good scientists) to treat the water with DEPC in the bottle it is going to be stored, hence eliminating all external sources of RNAse. Anyone care to comment?? And I don't see any reason why people should try to save a few minutes of their time and a few dollars and risk their expensive experiments. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of DK Sent: Wednesday, February 18, 2009 12:52 AM To: methods@magpie.bio.indiana.edu Subject: Re: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) In article , Michael Sullivan wrote: > >Another hint is that usually there is no special need to treat water >with DEPC. My experience is that 18 megaohm water from a milliQ system >is essentially RNase free. Of course. The water goes through several cartridges that all absorb proteins and peptides (ion exchange resins, activated charcoal and even the final 0.2 micron filter that is not designed to be low protein binding) plus the whole system is "sanitized" with NaOH on a periodic basis, killing any microbes that might be a source of RNAses. DK _______________________________________________ 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 nick.theodorakis from gmail.com Wed Feb 18 13:10:22 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Wed Feb 18 19:01:02 2009 Subject: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) References: Message-ID: On Feb 18, 11:55?am, "Jayakumar, R" wrote: > But RNAse contamination of the milliq water can happen when it is in > transit to your reaction tubes which may also be RNAse free, and also > the tubing which dispenses the water from the Milliq cartridge to your > "Rnase-free" storage container. ?It is important the glass bottles or > carboys or other plasticware that is used to store this milliQ water is > clean too, wherein lies the problem that it is virtually impossible to > eliminate RNAses during the transitionary phase. ?That is why it is > always PRUDENT (an important word in the vocabulary of good scientists) > to treat the water with DEPC in the bottle it is going to be stored, > hence eliminating all external sources of RNAse. ?Anyone care to > comment?? ?And I don't see any reason why people should try to save a > few minutes of their time and a few dollars and risk their expensive > experiments. My comment is the same as the others who note that ultra-pure water is free of RNase contamination and that RNAse-phobia is way overdone. I haven't used any DEPC or similar reagents since grad school, and haven't had a problem even when probing for full length mRNAs in Northerns. I never understood why anyone would pay good money to get their water ultrapure and then deliberately contiminate with something else. If your water is not pure enough to use for RNA, then it's probably not good for most other applications. I don't store water for RNA use, in any case; I get it fresh from the tap. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From mlsulliv from wisc.edu Wed Feb 18 16:03:11 2009 From: mlsulliv from wisc.edu (Michael Sullivan) Date: Wed Feb 18 19:01:08 2009 Subject: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) In-Reply-To: <97101976F8A044468CA74FE11883B90E173E7C3D@VISTA.roswellpark.org> References: <97101976F8A044468CA74FE11883B90E173E7C3D@VISTA.roswellpark.org> Message-ID: It seems to me there is far more "transitory" time in the course of an experiment over which you have little control than the brief time you will have a container open to dispense water from the milliQ into it. How long will a bottle or tube of DEPC treated water be open while you are later dispensing it into reaction tubes, how long are reaction tubes open while you are adding other reagents, etc? And how about all those other reagents that go into an experiment, many of which you cannot treat with DEPC? And DEPC treating the water because the container might not be clean enough seems extreme since glassware could be treated with dilute base or bleach or baked to render it RNase free, or one could use sterile disposable plasticware which is again, in my experience, RNase free whether marketed that way or not. Also, where do you draw the line at being "prudent"? Besides gloves, should you wear a surgical cap, gown and mask? Should you work in a hood or a glove box? In my experience, in a reasonably clean lab, there just isn't RNase all over the place ready to fall into your experiment and using MilliQ water directly without DEPC treatment has simply not been a problem for me or (judging from previous discussions here) many other researchers. So while I agree we shouldn't be penny wise and pound foolish, I don't have a problem saving my pennies by not spending them to solve a non-existent problem. Mike On Feb 18, 2009, at 10:55 AM, Jayakumar, R wrote: > > But RNAse contamination of the milliq water can happen when it is in > transit to your reaction tubes which may also be RNAse free, and also > the tubing which dispenses the water from the Milliq cartridge to your > "Rnase-free" storage container. It is important the glass bottles or > carboys or other plasticware that is used to store this milliQ > water is > clean too, wherein lies the problem that it is virtually impossible to > eliminate RNAses during the transitionary phase. That is why it is > always PRUDENT (an important word in the vocabulary of good > scientists) > to treat the water with DEPC in the bottle it is going to be stored, > hence eliminating all external sources of RNAse. Anyone care to > comment?? And I don't see any reason why people should try to save a > few minutes of their time and a few dollars and risk their expensive > experiments. > > Jay > > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu > [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of DK > Sent: Wednesday, February 18, 2009 12:52 AM > To: methods@magpie.bio.indiana.edu > Subject: Re: RNase Zap (Invitrogen) and RNase Away (Molecular > Bioproducts) > > In article , Michael > Sullivan wrote: >> >> Another hint is that usually there is no special need to treat water >> with DEPC. My experience is that 18 megaohm water from a milliQ >> system >> is essentially RNase free. > > Of course. The water goes through several cartridges that all absorb > proteins and peptides (ion exchange resins, activated charcoal and > even > the final 0.2 micron filter that is not designed to be low protein > binding) plus the whole system is "sanitized" with NaOH on a periodic > basis, killing any microbes that might be a source of RNAses. > > DK > > > > > _______________________________________________ > 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. > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods --- Michael L. Sullivan Plant Research Molecular Geneticist US Dairy Forage Research Center ARS-USDA 1925 Linden Drive West Madison, WI 53706 (608) 890-0046 (Phone) (608) 890-0076 (FAX) From whyharish from gmail.com Wed Feb 18 19:47:08 2009 From: whyharish from gmail.com (whyharish reddy) Date: Wed Feb 18 21:46:30 2009 Subject: Methods Digest, Vol 45, Issue 11 In-Reply-To: <200902161703.n1GH3m822030@net.bio.net> References: <200902161703.n1GH3m822030@net.bio.net> Message-ID: <5894aabe0902181647y697d4ebfpcb69b1d42064faa2@mail.gmail.com> Hi Thank you, for giving information on enzyme stability...! On Mon, Feb 16, 2009 at 10:33 PM, wrote: > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. Re: STABILIZERS FOR ENZYMES (mainly XYLANASE)Methods Digest, > Vol 45, Issue 7 (Dr Engelbert Buxbaum) > 2. Detergent concentration for nuclear membrane dissolving > (Werner Straube) > 3. methylcellulose plating problem (Scott Brown) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Sun, 15 Feb 2009 08:39:38 -0400 > From: "Dr Engelbert Buxbaum" > Subject: Re: STABILIZERS FOR ENZYMES (mainly XYLANASE)Methods Digest, > Vol 45, Issue 7 > To: methods@net.bio.net > Message-ID: > Content-Type: text/plain; format=flowed; delsp=yes; > charset=iso-8859-15 > > Am 11.02.2009, 15:29 Uhr, schrieb whyharish reddy : > > > *I am unable to maintain my enzyme activity not more than 2 days, * > > *please help in mainatining the activity of XYLANASE ENZYME for more than > > 2-3 months, suggest some Enzyme Stablizers?* > > Have you done a literature search (scholar.google.com, PubMed)? Of hand I > can help only with some general hints: > - Stability is temperature dependent. As is generally known, enzymes can > be heat-denatured, but some are also cold-denatured. > - Stability is strongly pH-dependent. Avoid storing enzymes near the pI, > and at extreme pH values. Other than that, trial and error. > - Some enzymes are sensitive to ionic strength and osmotic pressure. Keep > both near physiological values. For intracellular enzymes use KCl, for > extracellular NaCl to adjust ionic strenght. Manitol, sucrose, glycerol or > similar polyols are used to maintain osmotic pressure. > - Some enzymes require specific cofactors for activity and may be > stabilised in their presence. Mg, Zn... > - Sterile filter your preparation using a low-protein binding membrane > (0.22 ?m) and use aseptic techniques for handling. Add presevatives (Na > azide, Thimerosal...) but check that they are compatible with the enzyme. > - Check an inactive preparation for what actually happend, for example by > SDS-PAGE, light scattering and CD-spectroscopy. Is the enzyme degraded > (presence of proteolytic enzymes, microorganisms), forms aggregates or > changes conformation? That info can help you develop more targeted > approaches to the problem. > > > ------------------------------ > > Message: 2 > Date: Sun, 15 Feb 2009 21:29:53 +0100 > From: "Werner Straube" > Subject: Detergent concentration for nuclear membrane dissolving > To: > Message-ID: > <36B75E885C4376419EC0020DA151B41C01149A1B@msx.w2k.biochem.mpg.de> > Content-Type: text/plain; charset="us-ascii" > > Hello everybody, > > A friend of mine is interested isolating nuclear proteins. After he has > isolated the nuclei of cells he then wants to dissolve the nuclear > membrane without destroying the interactions of nuclear proteins. > > We were already discussing the type and concentrations of detergents, > however we did not get final clue. > > Can anyone help me out there with some suggestions or protocols or are > there good references (protocols) in the literature (for general > membrane dissolving and in particular nuclear membrane dissolving) ? > > Thank you very much, > > Werner > > > > > > ------------------------------ > > Message: 3 > Date: Mon, 16 Feb 2009 12:23:26 +1100 > From: "Scott Brown" > Subject: methylcellulose plating problem > To: > Message-ID: > <2A67EA781EC7F949A2AB0A0D07A86C6A02972C3D@mail01.ccia.local> > Content-Type: text/plain; charset="iso-8859-1" > > Hi, > > This may be a long shot but i am banging my head against the wall over it. > I have been doing some methocult plating, basically i take normal Nalm-6 > cells, add 1.6 mls methocult, 1.2 mls FCS, 1.2mls IMDM media and then i add > dexamethosone at a conc. of 8uM. > Last year this would kill the majority of the Nalm-6 cells (EC50 = 6nM), > there were always some colonies that would grow through so i added some > methotrexate to try and decrease the formation of colonies. > For the past 6 months I have been getting equal numbers of colonies in my > control (the nalms) as well as my experimental population of cells. I've > checked my conc. of dex, repeated MTTs to confirm the EC50, repeated the > plating several times but continue to get too many colonies being formed in > the control. Previous plating showed a distinct difference in the amount of > colonies formed in the control and the experimental situation. > > Does anyone have any ideas as to what may be going on or any suggestions > for an experiment i may be able to do to get to the bottom of this? > > cheers > > Scott > > Scott Brown > PhD Candidate > Molecular Carcinogenesis Program > Children's Cancer Institute of Australia for Medical Research > High Street (PO Box 81) > RANDWICK NSW 2031 > AUSTRALIA > > Phone: +61 2 9382 1829 > Fax: +61 2 9382 1850 > Email: sbrown@ccia.unsw.edu.au > Web: www.ccia.org.au > Children's Cancer Institute Australia is the only independent medical > research institute in Australia solely devoted to research into the causes, > prevention and cure of childhood cancer. Our vision is to save the lives of > all children with cancer and 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. > > Children's Cancer Institute Australia for Medical Research is the only > independent medical research institute in Australia devoted to research into > the causes, prevention, better treatment and ultimately a cure of childhood > cancer. Our vision is to save the lives of all children with cancer and to > eliminate their suffering. > > The information contained in this message and any annexure is confidential > and intended only for the named recipient(s). If you have received this > message in error please contact the sender immediately and destroy the > original message. > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 45, Issue 11 > *************************************** > -- Y.HARISH KUMAR REDDY From prae from gmx.net Thu Feb 19 04:11:35 2009 From: prae from gmx.net (Christian Praetorius) Date: Thu Feb 19 12:31:40 2009 Subject: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) References: <7jKml.1580$Sv4.243@newsfe15.iad> Message-ID: <704m27FmtselU1@mid.individual.net> dk@no.email.thankstospam.net (DK) wrote: >In my experience, with autoclaves being dirty, dirty things, anything >autoclaved is simply not clean. Sterile - yes, but not clean. Few Especially, when the same autoclave is used for autoclaving trash and clean things. >lab it is a rule that protein crystallization has to be done with tips >out of the bag, never to be autoclaved. These tips (of course not touched with bare fingers) should be free of everything because of the production process. Christian -- X-no-Sig: yes From aawara from pontiff-playground.org Thu Feb 19 04:39:55 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Thu Feb 19 12:31:48 2009 Subject: RNase Zap (Invitrogen) and RNase Away (Molecular Bioproducts) References: <7jKml.1580$Sv4.243@newsfe15.iad> Message-ID: In , DK wrote: > Aawara, I don't doubt your observation for a nanosecond but let > me ask this: Any chance that the RNAse activity was there > *because* of the autoclaving? 100% possible. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From editor from gene-quantification.info Thu Feb 19 06:04:47 2009 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Thu Feb 19 12:36:15 2009 Subject: qPCR NEWS January 2009 - update in available real-time PCR cycler Message-ID: qPCR NEWS January 2009 - update in available real-time PCR cycler ---------------------------------------------------------------------------= ------------- 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: - qPCR 2009 Event =3D> http://www.qPCR2009.net - we are proud to present 230 scientific contributions (86 talks & 144 posters) have a look on the preliminary online agenda =3D> http://online-AGENDA.qpcr2009.net/ - real-time PCR Cycler page updated =3D> http://cyclers.gene-quantification= .info/ - webinar page updated =3D> http://webinar.gene-quantification.info/ - Online translation service of the Gene Quantification =3D> http://translation.gene-quantification.info/ New qPCR workshop modules at the TATAA Biocenter Germany =3D> http://tataa.gene-quantification.info/ European wide qPCR application workshops =3D> register now ! =3D> course program spring - summer 2009 =3D> http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pd= f ---------------------------------------------------------------------------= ----- real-time PCR Cycler On this page the most prominent real-time PCR cycler are described. In the cycler descriptions the specifications and the advantages of the displayed systems are shown. Which real-time platform meets your requirements best, depends on your research application. Some of the systems are designed for research with low capacities and others are for high-throughput applications, most in combination with pipetting robots. Most of them use solid-block for thermal cycling, other use hot- and cooled-air. Most differences are obviously in the application software, especially in the way of data analysis and how the derived crossing points or threshold levels are computed. Each of these systems employs either one of several general types of fluorescent probes for detection. There are also big differences how data are displayed. Some of the limitations of end-point detection in (RT-) PCR have been assuaged in real-time PCR systems, a number of which are now on the market. These systems offer many general technical advantages, including reduced probabilities of variability and contamination, as well as online monitoring and the lack of need for post reaction analyses. Further, some of these systems were developed with contemporary applications such as quantitative PCR, multiplexing, and high-throughput analysis in mind. Initial template levels can be calculated by analysing the shape of the curve or by determining when the signal rises above some threshold value. Several commercially available real-time PCR systems are overviewed on this page and/or summarized in the accompanying table. =3D> http://cyclers.gene-quantification.info/ ---------------------------------------------------------------------------= ----- Real-Time PCR: Current Technology and Applications http://www.horizonpress.com/realtimepcr Editor: Julie Logan, Kirstin Edwards and Nick Saunders Applied and Functional Genomics, Health Protection Agency, London (2009) ISBN: 9781904455394 Publisher: Caister Academic Press Chapter 4 - Reference Gene Validation Software for Improved Normalization J. Vandesompele, M. Kubista and M. W. Pfaffl (2009) http://www.gene-quantification.de/Vandesompele-Kubista-Pfaffl-real-time-PCR= -chapter-4.pdf Real-time PCR is the method of choice for expression analysis of a limited number of genes. The measured gene expression variation between subjects is the sum of the true biological variation and several confounding factors resulting in non-specific variation. The purpose of normalization is to remove the non-biological variation as much as possible. Several normalization strategies have been proposed, but the use of one or more reference genes is currently the preferred way of normalization. While these reference genes constitute the best possible normalizers, a major problem is that these genes have no constant expression under all experimental conditions. The experimenter therefore needs to carefully assess whether a certain reference gene is stably expressed in the experimental system under study. This is not trivial and represents a circular problem. Fortunately, several algorithms and freely available software have been developed to address this problem. This chapter aims to provide an overview of the different concepts. Chapter 5 - Data Analysis Software M. W. Pfaffl, J. Vandesompele and M. Kubista (2009) http://www.gene-quantification.de/Pfaffl-Kubista-Vandesompele-real-time-PCR= -chapter-5.pdf Quantitative real-time RT-PCR (qRT-PCR) is widely and increasingly used in any kind of mRNA quantification, because of its high sensitivity, good reproducibility and wide dynamic quantification range. While qRT-PCR has a tremendous potential for analytical and quantitative applications, a comprehensive understanding of its underlying principles is important. Beside the classical RT-PCR parameters, e.g. primer design, RNA quality, RT and polymerase performances, the fidelity of the quantification process is highly dependent on a valid data analysis. This review will cover all aspects of data acquisition (trueness, reproducibility, and robustness), potentials in data modification and will focus particularly on relative quantification methods. Furthermore useful bioinformatical, biostatical as well as multi-dimensional expression software tools will be presented. ---------------------------------------------------------------------------= ----- Webinars in qPCR & RNAinterference & Molecular-Biology Here you find a huge summary of webinars in the field of qPCR & RNAinterference & Molecular-Biology =3D> http://webinar.gene-quantification.info/ ---------------------------------------------------------------------------= ----- online translation Since October 2008 we provide an online translation service of the Gene Quantification pages to several languages. Please recognize this is an automatic and robotic based translation service, and therefore we can give NO guarantee about the generated content. It should help to understand the "rough" content of the Gene Quantification pages, but still the original is the ENGLISH version: http://translation.gene-quantification.info/ http://www.gene-quantification.asia http://chinese.gene-quantification.asia/ http://dutch.gene-quantification.info/ http://french.gene-quantification.info/ http://german.gene-quantification.info/ http://greek.gene-quantification.info/ http://italian.gene-quantification.info/ http://japanese.gene-quantification.asia/ http://korean.gene-quantification.asia/ http://portuguese.gene-quantification.info/ http://russian.gene-quantification.asia/ http://spanish.gene-quantification.info/ ---------------------------------------------------------------------------= ----- With the new qPCR INFO PORTAL and all the presented tools we will help you with to find the right information about qPCR and related topics in Molecular Biology in the literature and in the World Wide Web. =3D> Papers / Protocols / Methods / Databases / Alets / Feeds / Books / Forums / E-mail / Directory http://infoportal.gene-quantification.info/ ---------------------------------------------------------------------------= ----- Upcoming Events World-wide academic and commercial qPCR Events http://events.gene-quantification.info/ Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars, qPCR Education Program, etc. Please submit your qPCR event here =3D> events@gene- quantification.info ---------------------------------------------------------------------------= ----- qPCR 2009 EVENT - 9 - 13 March 2009 more news here =3D> http://www.qPCR2009.net download event FLYER =3D> http://www.bioeps.com/qpcr2009/qPCR-2009-3rd-an= nouncement.pdf We are proud to present 230 scientific contributions (86 talks & 144 posters) have a look on the preliminary online agenda =3D> http://online-AGENDA.qpcr2009.net/ It is a pleasure to announce the Nobel Prize Laureate Kary Mullis at the qPCR 2009 event in an own session =9325th Anniversary of PCR=94 List of confirmed speakers =3D> http://speakers.qpcr2009.net/ An industrial exhibition with the 30 world leading companies will be held during the qPCR Symposium March 9-11 =3D> http://exhibition.qpcr2009= .net/ Our sponsors =3D> http://sponsors.qpcr2009.net/ Please register until 31st December to get the early registartion fee =3D> http://registration.qpcr2009.net/ Keynote lectures The Pioneer in PCR Kary B. Mullis 1993 Nobel Prize in Chemistry - 25th Anniversary of PCR Stephen A. Bustin Professor of Molecular Science, Institute of Cell and Molecular Science, Queen Mary's School of Medicine and Dentistry, University of London, UK - A new qPCR assay for the detection of Clostridium difficile - MIQE- guidelines for publication of qPCR data Ken Livak Senior Scientific Fellow, Fluidigm Corporation,San Francisco, CA, US - Moving from qPCR Assays to qPCR Arrays. ---------------------------------------------------------------------------= ----- qPCR WORKSHOP BioEPS GmbH / TATAA Biocenter Germany - qPCR Application workshops At the TATAA Biocenter Germany we offer qPCR application workshops, a 3-day qPCR Core Module and a 2-day qPCR Biostatistics Module. All courses are held regularly in G=F6teborg, Sweden, in English and in Freising-Weihenstephan, Germany, in German and English, and in Prague, Czech Republic in English and Czech. Depending on the occasion the workshop language and the different prices may apply. Further customized workshops and specialized trainings will be held as well across Europe and world-wide. TATAA Biocenter Germany workshops are held in cooperation with BioEPS GmbH, located at the campus of the Technical University of Munich, in Freising-Weihenstephan, very close to the Munich Airport (MUC). For more information and registration, please see our web page: =3D> http://TATAA.gene-quantification.info/ Course Occasions 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 27 - 29 April 2009 (E) NEW singel-cell qPCR Application Workshop 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.) =3D> http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pd= f ---------------------------------------------------------------------------= ----- Forward Please send the qPCR NEWS to further scientists and friends who are interested in qPCR ! Best regards, Michael W. Pfaffl responsible Editor of the Gene Quantification Pages http://www.gene-quantification.info ---------------------------------------------------------------------------= ----- If this newsletter is not displayed correctly by your email client, please use following link: http://qpcrnews.gene-quantification.info/ The qPCR NEWS and the Gene Quantification Pages are educational sites with the only purpose of facilitating access to qPCR related information on the internet. The qPCR NEWS and the Gene Quantification Pages are edited by Michael W. Pfaffl. Copyright =A9 2005 - 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 The qPCR newsletter was end to [alle-adressen] To subscribe or change your e-mail address in qPCR NEWS, and if you would like to receive future issues FREE of charge, please send an e- mail with the subject SUBSCRIBE to mailto:newsletter@gene- quantification.info?subject=3DSUBSCRIBE From Mozella.Moore from carolinashealthcare.org Fri Feb 20 12:30:52 2009 From: Mozella.Moore from carolinashealthcare.org (Moore, Mozella) Date: Fri Feb 20 13:48:40 2009 Subject: RNA compatible with isopentane? Message-ID: <23AFBE9BE2565F44A19D448AE6EEA1AF1C5D68F6@CHS-XCHG-VS-05.Carolinas.org> Hello Brad, I have performed traditional frozen methods utilizing OCT for freezing tissue in clinical setting. The research protocol requires a large volume and rapid freezing method to store animal tissue for future usage. Will you please email a copy of protocol if available? I am familiar with procedure, but would like to have protocol for technical or trouble shooting if necessary. Thanks, Mozella Moore Cannon Research Center 704-355-8202 ----------------------------------------- This electronic message may contain information that is confidential and/or legally privileged. It is intended only for the use of the individual(s) and entity named as recipients in the message. If you are not an intended recipient of this message, please notify the sender immediately and delete the material from any computer. Do not deliver, distribute or copy this message, and do not disclose its contents or take any action in reliance on the information it contains. Thank you. From sissi_2009 from hotmail.com Fri Feb 20 12:42:02 2009 From: sissi_2009 from hotmail.com (Sissi zhang) Date: Fri Feb 20 13:48:52 2009 Subject: E. coli DH5 alpha culture problem Message-ID: Dear Members=2C I need quite a bit of pUC19 vector=2C so I transformed it into DH5 alpha an= d grew the culture. I run into a problem with DH5 alpha culture. The growth= rate seemed low. I did not get much pUC19 DNA out of 500 mL culture. Here are the things I observed: 1) Transformation efficiency was good=2C reached 10^7=2C however=2C the col= onies were very small after overnight growth on selective LB Miller agar pl= ate (less than 0.5 mm in diameter) 2) Starter culture gave OD600=3D0.5 after 8 hours growth (a single colony i= n 2 mL LB=2C Miller broth with 100 ug/mL ampicillin) 3) 2nd culture: 1 mL of starter into 500 mL LB Miller broth with ampicillin OD600=3D0.9 after 12 hours growth OD600=3D1.15 after 16 hours growth OD600=3D1.34 after 18 hours growth 4) Cell pellet wet weight after 18 hours growth was 1 g from 500 mL culture= . The growth conditions I used:=20 1)500 mL LB Miller broth in 2-L flask 2) Shaking at 37 C at 300 rpm 3) 100 ug/mL ampicilin in both LB agar plate and LB broth Questions: 1) Do you think the growth rate was slow? 2) If so=2C what could be the reasons? I heard that DH5 alpha tend to give = small colonies=2C even so=2C why the total yield was so low? I guess I coul= d let the colonies continually grow to much bigger colonies before liquid c= ulture. If I start with a much bigger colony=2C would I get OD600=3D4.5 aft= er 16 hours growth(this growth rate is shown in Qiagen plasmid DNA purifica= tion brochure)? 3) pUC19 should be high copy vector but I got only 0.6 mg DNA out of 500 mL= culture I know there are a lot of experts on this topic. Please help! Thank you=2C Sissi _________________________________________________________________ Windows Live=99: E-mail. Chat. Share. Get more ways to connect.=20 http://windowslive.com/online/hotmail?ocid=3DTXT_TAGLM_WL_HM_AE_Faster_0220= 09= From shifalich from rediffmail.com Sat Feb 21 12:50:41 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Sat Feb 21 13:49:37 2009 Subject: E. coli DH5 alpha culture problem Message-ID: <1235159283.S.4403.11701.f4mail-235-204.rediffmail.com.old.1235238641.16613@webmail.rediffmail.com> On Sat, 21 Feb 2009 01:18:03 +0530 wrote > > > > > > > >Dear Members, > >I need quite a bit of pUC19 vector, so I transformed it into DH5 alpha and grew the culture. I run into a problem with DH5 alpha culture. The growth rate seemed low. I did not get much pUC19 DNA out of 500 mL culture. > >Here are the things I observed: >1) Transformation efficiency was good, reached 10^7, however, the colonies were very small after overnight growth on selective LB Miller agar plate (less than 0.5 mm in diameter) Can you please tell the amount of transformation reaction plated? Also, is that you plated everything? I have successfully got nearly 1mm colonies with DH5 alpha! >2) Starter culture gave OD600=0.5 after 8 hours growth (a single colony in 2 mL LB, Miller broth with 100 ug/mL ampicillin) >3) 2nd culture: 1 mL of starter into 500 mL LB Miller broth with ampicillin > ? ?OD600=0.9 after 12 hours growth > ? ?OD600=1.15 after 16 hours growth > ? ?OD600=1.34 after 18 hours growth >4) Cell pellet wet weight after 18 hours growth was 1 g from 500 mL culture. > >The growth conditions I used: >1)500 mL LB Miller broth in 2-L flask Can you please tell the composition of LB miller ?( Make sure you read the one written on the bottle from where you took the powder to make medium) >2) Shaking at 37 C at 300 rpm >3) 100 ug/mL ampicilin in both LB agar plate and LB broth > >Questions: >1) Do you think the growth rate was slow? >2) If so, what could be the reasons? I heard that DH5 alpha tend to give small colonies, even so, why the total yield was so low? I guess I could let the colonies continually grow to much bigger colonies before liquid culture. If I start with a much bigger colony, would I get OD600=4.5 after 16 hours growth(this growth rate is shown in Qiagen plasmid DNA purification brochure)? >3) pUC19 should be high copy vector but I got only 0.6 mg DNA out of 500 mL culture > >I know there are a lot of experts on this topic. Please help! > >Thank you, > >Sissi > > > >_________________________________________________________________ >Windows Live: E-mail. Chat. Share. Get more ways to connect. >http://windowslive.com/online/hotmail?ocid=TXT_TAGLM_WL_HM_AE_Faster_022009_______________________________________________ >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 peter.ianakiev from gmail.com Tue Feb 24 08:54:37 2009 From: peter.ianakiev from gmail.com (peter) Date: Tue Feb 24 12:01:42 2009 Subject: E. coli DH5 alpha culture problem References: Message-ID: <2593f932-192a-485e-9e32-cb4ec4bf5d9c@l16g2000yqo.googlegroups.com> On Feb 20, 12:42=A0pm, Sissi zhang wrote: > Dear Members, > > I need quite a bit of pUC19 vector, so I transformed it into DH5 alpha an= d grew the culture. I run into a problem with DH5 alpha culture. The growth= rate seemed low. I did not get much pUC19 DNA out of 500 mL culture. > > Here are the things I observed: > 1) Transformation efficiency was good, reached 10^7, however, the colonie= s were very small after overnight growth on selective LB Miller agar plate = (less than 0.5 mm in diameter) > 2) Starter culture gave OD600=3D0.5 after 8 hours growth (a single colony= in 2 mL LB, Miller broth with 100 ug/mL ampicillin) > 3) 2nd culture: 1 mL of starter into 500 mL LB Miller broth with ampicill= in > =A0 =A0 OD600=3D0.9 after 12 hours growth > =A0 =A0 OD600=3D1.15 after 16 hours growth > =A0 =A0 OD600=3D1.34 after 18 hours growth > 4) Cell pellet wet weight after 18 hours growth was 1 g from 500 mL cultu= re. > > The growth conditions I used: > 1)500 mL LB Miller broth in 2-L flask > 2) Shaking at 37 C at 300 rpm > 3) 100 ug/mL ampicilin in both LB agar plate and LB broth > > Questions: > 1) Do you think the growth rate was slow? > 2) If so, what could be the reasons? I heard that DH5 alpha tend to give = small colonies, even so, why the total yield was so low? I guess I could le= t the colonies continually grow to much bigger colonies before liquid cultu= re. If I start with a much bigger colony, would I get OD600=3D4.5 after 16 = hours growth(this growth rate is shown in Qiagen plasmid DNA purification b= rochure)? > 3) pUC19 should be high copy vector but I got only 0.6 mg DNA out of 500 = mL culture > > I know there are a lot of experts on this topic. Please help! > > Thank you, > > Sissi > > _________________________________________________________________ > Windows Live=99: E-mail. Chat. Share. Get more ways to connect.http://win= dowslive.com/online/hotmail?ocid=3DTXT_TAGLM_WL_HM_AE_Faster_... Well, you have to seed pre-culture single colony in say 5-10 ml LB +Abt, and then innoculate the 500 flask. my2c From virashkgupta from gmail.com Tue Feb 24 08:06:14 2009 From: virashkgupta from gmail.com (Virash Gupta) Date: Tue Feb 24 12:01:49 2009 Subject: Methods Digest, Vol 45, Issue 17 In-Reply-To: <200902221705.n1MH5A117910@net.bio.net> References: <200902221705.n1MH5A117910@net.bio.net> Message-ID: Some points need to be clarified. starter culture (if it is DH5 ?)- you have been addding ampicillin which inhibited growth. It is also possible that puc 19 is defective for gene that controls high copy number. Acquire puc 19 from some other souce or try with some other plasmid. This will help in standardizing transformation conditions. Streak DH5 culture on LB agar, isolate well growing colonies and streak on LB and LB ampicillin plates. Select clones that are growing on LB and not on LB ampicillin for transformation. Hope it will solve. On 2/22/09, 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: E. coli DH5 alpha culture problem (shifali chatrath) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: 21 Feb 2009 17:50:41 -0000 > From: "shifali chatrath" > Subject: Re: E. coli DH5 alpha culture problem > To: > Cc: methods@magpie.bio.indiana.edu > Message-ID: > < > 1235159283.S.4403.11701.f4mail-235-204.rediffmail.com.old.1235238641.16613@webmail.rediffmail.com > > > > Content-Type: text/plain; charset="ISO-8859-1" > > > > On Sat, 21 Feb 2009 01:18:03 +0530 wrote > > > > > > > > > > > > > > > >Dear Members, > > > >I need quite a bit of pUC19 vector, so I transformed it into DH5 alpha and > grew the culture. I run into a problem with DH5 alpha culture. The growth > rate seemed low. I did not get much pUC19 DNA out of 500 mL culture. > > > >Here are the things I observed: > >1) Transformation efficiency was good, reached 10^7, however, the colonies > were very small after overnight growth on selective LB Miller agar plate > (less than 0.5 mm in diameter) > > Can you please tell the amount of transformation reaction plated? Also, is > that you plated everything? > > I have successfully got nearly 1mm colonies with DH5 alpha! > > >2) Starter culture gave OD600=0.5 after 8 hours growth (a single colony in > 2 mL LB, Miller broth with 100 ug/mL ampicillin) > > >3) 2nd culture: 1 mL of starter into 500 mL LB Miller broth with > ampicillin > > OD600=0.9 after 12 hours growth > > OD600=1.15 after 16 hours growth > > OD600=1.34 after 18 hours growth > > > >4) Cell pellet wet weight after 18 hours growth was 1 g from 500 mL > culture. > > > >The growth conditions I used: > >1)500 mL LB Miller broth in 2-L flask > > Can you please tell the composition of LB miller ?( Make sure you read the > one written on the bottle from where you took the powder to make medium) > > >2) Shaking at 37 C at 300 rpm > >3) 100 ug/mL ampicilin in both LB agar plate and LB broth > > > >Questions: > >1) Do you think the growth rate was slow? > >2) If so, what could be the reasons? I heard that DH5 alpha tend to give > small colonies, even so, why the total yield was so low? I guess I could let > the colonies continually grow to much bigger colonies before liquid culture. > If I start with a much bigger colony, would I get OD600=4.5 after 16 hours > growth(this growth rate is shown in Qiagen plasmid DNA purification > brochure)? > >3) pUC19 should be high copy vector but I got only 0.6 mg DNA out of 500 > mL culture > > > >I know there are a lot of experts on this topic. Please help! > > > >Thank you, > > > >Sissi > > > > > > > >_________________________________________________________________ > >Windows Live: E-mail. Chat. Share. Get more ways to connect. > > > http://windowslive.com/online/hotmail?ocid=TXT_TAGLM_WL_HM_AE_Faster_022009_______________________________________________ > >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 > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 45, Issue 17 > *************************************** > -- 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 stxsz1 from nottingham.ac.uk Wed Feb 25 11:29:14 2009 From: stxsz1 from nottingham.ac.uk (Zhong Silin) Date: Wed Feb 25 14:00:51 2009 Subject: 2D gel or MS? for comparison proteomic Message-ID: <5D7E3A8B871FD645A3A9FB96F3951EF8433224@VUIEXCHA.ad.nottingham.ac.uk> Hi 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. Thanks in advance SZ 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 Thu Feb 26 03:35:31 2009 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu Feb 26 10:39:31 2009 Subject: Regarding Plasmid Expression Message-ID: Dear all,I have been working with a gene cloned into pcDNA 3.1 V5/His vector. However, over time the expression has decreased drastically. However, another gene cloned into same vector has consistently high expression, till now (that rules out cell problems too). I did prepare fresh Midi stock..but problem remains. What has gone wrong? faithfully sudheendra. -- Think before agree Think before you nod STOP thinking to be a God From shifalich from rediffmail.com Thu Feb 26 07:56:45 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Thu Feb 26 10:40:11 2009 Subject: 2D gel or MS? for comparison proteomic Message-ID: <1235588605.S.3954.56551.f4mail-235-218.rediffmail.com.1235653005.41589@webmail.rediffmail.com> Please send me the paper where in they use 2D and MS/MS techniques to quantitate the level of proteins ! 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.If you want to compare quantitatively, simply running SDS PAGE with normalized protein loading should serve the purpose and if you particularly want to compare two known proteins, SDS PAGE followed by western will help! Shifali On Thu, 26 Feb 2009 00:33:25 +0530 wrote >Hi > >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. > >Thanks in advance >SZ > >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. > >_______________________________________________ >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 huazhongw from hotmail.com Thu Feb 26 09:32:50 2009 From: huazhongw from hotmail.com (huazhongw) Date: Thu Feb 26 10:46:57 2009 Subject: vector ligation problem References: <200902241705.n1OH5Re28059@net.bio.net> Message-ID: Dear memebers, I am working with vector constuction. I inserted a fragment into a vector by routine single-digest \electrophoresis\recovery\ligation\tranformation method. When I recut recombinant plasmids with the same enzyme, I run into a problem that the vector fragment size become smaller than the original cut vector. I don't know why and need help! Huazhong Wang From sudhee26 from gmail.com Thu Feb 26 10:57:15 2009 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu Feb 26 12:27:06 2009 Subject: vector ligation problem In-Reply-To: References: <200902241705.n1OH5Re28059@net.bio.net> Message-ID: Hi ,Check for the star activity of the enzyme you are using. Also following links might be useful to you. http://catalog.takara-bio.co.jp/en/product/basic_info.asp?unitid=U100005230 http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/cuteffects.html regards Sudheendra. On Thu, Feb 26, 2009 at 8:02 PM, huazhongw wrote: > > Dear memebers, > I am working with vector constuction. I inserted a fragment into a > vector by routine single-digest > \electrophoresis\recovery\ligation\tranformation method. When I recut > recombinant plasmids with the same enzyme, I run into a problem that the > vector fragment size become smaller than the original cut vector. > I don't know why and need help! > > > Huazhong Wang > > > _______________________________________________ > 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 asidhu from biomap.org Fri Feb 27 00:49:32 2009 From: asidhu from biomap.org (Amandeep Sidhu) Date: Fri Feb 27 08:47:05 2009 Subject: CFP: 4th Special Track on Ontologies for Biomedical Systems at 22nd IEEE CBMS 2009 Message-ID: <2dbc82b00902262149y1879f48dp49d64012cc099732@mail.gmail.com> CALL FOR PAPERS ---------- 4th Special Track on Ontologies for Biomedical Systems 3-4 August 2009 Albuquerque, New Mexico http://cbms09.biomap.org/ 22nd IEEE International Symposium on Computer-Based Medical Systems http://cvial.ee.ttu.edu/cbms2009/ ---------- Biomedical Ontologies have developed in an uncoordinated way, often reflecting mere relations of 'association' between what are called 'concepts', and serving primarily the purposes of information extraction from on-line biomedical literature and databases. In recent years, we have learned a great deal about the criteria which must be satisfied if ontology is to allow true information integration and automatic reasoning across data and information derived from different sources. The goal of this track is to survey existing biomedical ontologies and reform them in such a way as to allow true information integration in biomedical domain. Authors are invited to submit original papers exploring the theories, techniques, and applications of biomedical ontologies. Papers are invited (but not limited) to the following themes: * Biomedical Ontologies for Genetics, Proteomics, Diseases, Privacy etc * Conceptual Models for Biological and Medical Data * Semantics in Biological Data Modeling * Use of semantics to manage Interoperation in Biomedical Databases * Semantic Web technologies and formalisms for Biomedical Data * Ontology representation and exchange languages for bioinformatics * Biomedical Ontologies and OWL * Biological Data Integration and Management using Ontologies * Biomedical Data Engineering using Ontologies * Application of Biomedical Ontologies for Heterogeneous Database Access * Query Optimization Techniques for Biomedical Database using Ontologies * Support of Ontologies for Biological Information Retrieval and Web Services * Change Management in Biomedical Ontologies * Tools for Development and Management of Biomedical Ontologies PAPER SUBMISSION: No hardcopy submissions are being accepted. Electronic submissions of original technical research papers will only be accepted in PDF format. Use a maximum of 6 pages IEEE two-column format, including figures and references. All submissions will be done electronically via the CBMS 2009 web submission system ( http://www.easychair.org/conferences/?conf=ieeecbms2009). Select the track "Ontologies for Biomedical Systems", provide the information about the paper title, authors, keywords, and corresponding author's information (telephone, fax, mailing address, e-mail address). Please note that author names should not appear on the paper. Submission implies the willingness of at least one of the authors to register and present the paper at the CBMS 2009 Symposium. All papers will be peer reviewed by at least two independent referees. All accepted papers will be included in the conference proceedings. Selected papers from the track will be published in Special Issue of International Journal of Healthcare Information Systems and Informatics (IJHISI) in 2010. IMPORTANT DATES: April 1, 2009 Paper Submission Due May 25, 2009 Notification of acceptance June 21, 2009 Final camera-ready paper due June 21, 2009 Pre-registration deadline You must pre-register to have your paper published in the proceedings. If you only plan to attend and are not submitting a paper, pre-registration is still strongly encouraged. TRACK CHAIRS: Amandeep S. Sidhu WA Centre for Comparative Genomics, Murdoch University, Perth, Australia Matthew I. Bellgard WA Centre for Comparative Genomics, Murdoch University, Perth, Australia Jake Chen Indiana University - Purdue University, Indianapolis, USA For further questions, please contact technical program chair at: cbms@biomap.org ---------- From eenigoy from gmail.com Fri Feb 27 01:30:06 2009 From: eenigoy from gmail.com (yoginee budhkar) Date: Fri Feb 27 08:47:16 2009 Subject: plasmid isolation by salting out Message-ID: <77ddf6c70902262230i42df19ebj8b251c5bb9f9781c@mail.gmail.com> Dear All, There is a very routine and simple protocol for DNA isolation from whole blood by salting out with saturated NaCl, will it be equally effective to salt out plasmid DNA from bacteria? As in, if we used the first three steps of resuspension with glucose tris EDTA, alkaline lysis with SDS and NaOH and neutralisation with K acetate in glacial acetic acid, in stead of going for spin column or phenol chloroform or direct precipitation with alcohol, can we use the salting out protocol to get good quality plasmid? Regards, -- Yg From dandansnu from gmail.com Mon Feb 23 11:51:05 2009 From: dandansnu from gmail.com (Dan Liu) Date: Fri Feb 27 08:47:22 2009 Subject: Tris-Acetate SDS-PAGE gels Message-ID: From marguerite.b.colas from gsk.com Fri Feb 27 06:44:56 2009 From: marguerite.b.colas from gsk.com (marguerite.b.colas@gsk.com) Date: Fri Feb 27 08:47:50 2009 Subject: Recombinant protein precipitating upon freezing Message-ID: Hello Peter! I work at a pharmaceutical company and have been working on this study that involves freezing hemoglobin proteins, Every time we thaw our samples we notice that they go cloudy. At first we thought that we had been mixing our cell membrane pellet with the cell content supernatant. However, the time we took extra care to distinguish the supernatant from the pellet, we still noticed that our samples went cloudy after thawing them from the freezer. All our samples are kept in a -20c freezer. If you think you might know what might be happening to our samples, please let us know! thank you very much for your attention! ----------------------------------------------------------- This e-mail was sent by GlaxoSmithKline Services Unlimited (registered in England and Wales No. 1047315), which is a member of the GlaxoSmithKline group of companies. The registered address of GlaxoSmithKline Services Unlimited is 980 Great West Road, Brentford, Middlesex TW8 9GS. ----------------------------------------------------------- From pow.joshi from gmail.com Fri Feb 27 13:28:48 2009 From: pow.joshi from gmail.com (Pow Joshi) Date: Fri Feb 27 13:50:18 2009 Subject: Recombinant protein precipitating upon freezing In-Reply-To: References: Message-ID: <710764ea0902271028k7b80f9f2n956618557040232e@mail.gmail.com> 2009/2/27 > Hello Peter! > > I work at a pharmaceutical company and have been working on this study > that involves freezing hemoglobin proteins, Every time we thaw our samples > we notice that they go cloudy. > At first we thought that we had been mixing our cell membrane pellet with > the cell content supernatant. However, the time we took extra care to > distinguish the supernatant from the pellet, we still noticed that our > samples went cloudy after thawing them from the freezer. All our samples > are kept in a -20c freezer. > > If you think you might know what might be happening to our samples, please > let us know! methinks, you could try adding extra salt to the supernatant; or check the pH .... these are Fe bound proteins are'nt they?, perhaps if you need to have near neutral pH? ... I wonder if the Fe is forming hydroxides and precipitation out ...., in which case, you could add a bit of acid and resolublize? ....... Dima, may be you will be able to answer if I'm completely off the track? Pow > > > thank you very much for your attention! > ----------------------------------------------------------- > This e-mail was sent by GlaxoSmithKline Services Unlimited > (registered in England and Wales No. 1047315), which is a > member of the GlaxoSmithKline group of companies. The > registered address of GlaxoSmithKline Services Unlimited > is 980 Great West Road, Brentford, Middlesex TW8 9GS. > ----------------------------------------------------------- > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From davidminde from gmail.com Fri Feb 27 14:20:15 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Fri Feb 27 16:45:40 2009 Subject: Recombinant protein precipitating upon freezing In-Reply-To: <710764ea0902271028k7b80f9f2n956618557040232e@mail.gmail.com> References: <710764ea0902271028k7b80f9f2n956618557040232e@mail.gmail.com> Message-ID: <123243900902271120i10d4c96au1099735a99a59f10@mail.gmail.com> ... protein people dont keep their "pets" at -20 ... (better snapfreeze&store-80C or keep in lN2) David 2009/2/27, Pow Joshi : > 2009/2/27 > >> Hello Peter! >> >> I work at a pharmaceutical company and have been working on this study >> that involves freezing hemoglobin proteins, Every time we thaw our samples >> we notice that they go cloudy. >> At first we thought that we had been mixing our cell membrane pellet with >> the cell content supernatant. However, the time we took extra care to >> distinguish the supernatant from the pellet, we still noticed that our >> samples went cloudy after thawing them from the freezer. All our samples >> are kept in a -20c freezer. >> >> If you think you might know what might be happening to our samples, please >> let us know! > > > > methinks, you could try adding extra salt to the supernatant; or check the > pH .... these are Fe bound proteins are'nt they?, perhaps if you need to > have near neutral pH? ... I wonder if the Fe is forming hydroxides and > precipitation out ...., in which case, you could add a bit of acid and > resolublize? ....... Dima, may be you will be able to answer if I'm > completely off the track? > > Pow > >> >> >> thank you very much for your attention! >> ----------------------------------------------------------- >> This e-mail was sent by GlaxoSmithKline Services Unlimited >> (registered in England and Wales No. 1047315), which is a >> member of the GlaxoSmithKline group of companies. The >> registered address of GlaxoSmithKline Services Unlimited >> is 980 Great West Road, Brentford, Middlesex TW8 9GS. >> ----------------------------------------------------------- >> _______________________________________________ >> Methods mailing list >> Methods@net.bio.net >> http://www.bio.net/biomail/listinfo/methods >> > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 mobile phone +31(0)631154267 private address: Griftkade 4 bis 3572 TW Utrecht Science is what happens while we are making other plans (~John Lennon) From legatekBLAH from hotmail.com Sat Feb 28 03:09:52 2009 From: legatekBLAH from hotmail.com (Kyle Legate) Date: Sat Feb 28 14:30:30 2009 Subject: E. coli DH5 alpha culture problem In-Reply-To: <2593f932-192a-485e-9e32-cb4ec4bf5d9c@l16g2000yqo.googlegroups.com> References: <2593f932-192a-485e-9e32-cb4ec4bf5d9c@l16g2000yqo.googlegroups.com> Message-ID: <70s9pjFhv9gvU1@mid.individual.net> peter wrote: > Well, you have to seed pre-culture single colony in say 5-10 ml LB > +Abt, and then innoculate the 500 flask. > my2c I agree, the seeding culture was probably too small and not ready to add to the 500 mL culture, resulting in an unacceptably long lag phase. It should be 5-10 mL as Peter said and you should not be able to see through it when you add to the larger flask. Some people spin down the starter culture and resuspend in fresh LB-Amp to remove the secreted beta-lactamase but personally I don't do this for plasmid amplification. The long lag phase you experienced due to a dilute seed culture containing beta-lac may have depleted the antibiotic, resulting in some of the bacteria losing the plasmid, resulting in low yield. For a high yield of plasmid you can grow DH5a in Superbroth: 32g tryptone, 20g yeast extract, 5g NaCl, 1.25mL 4M NaOH per litre. I routinely get >1 mg of DNA per 100 mL of culture with this recipe. From legatekBLAH from hotmail.com Sat Feb 28 03:47:43 2009 From: legatekBLAH from hotmail.com (Kyle Legate) Date: Sat Feb 28 14:31:10 2009 Subject: Recombinant protein precipitating upon freezing In-Reply-To: References: Message-ID: <70sc0hFi0amgU1@mid.individual.net> marguerite.b.colas@gsk.com wrote: > Hello Peter! > > I work at a pharmaceutical company and have been working on this study > that involves freezing hemoglobin proteins, Every time we thaw our samples > we notice that they go cloudy. > At first we thought that we had been mixing our cell membrane pellet with > the cell content supernatant. However, the time we took extra care to > distinguish the supernatant from the pellet, we still noticed that our > samples went cloudy after thawing them from the freezer. All our samples > are kept in a -20c freezer. > Is there a reason you're keeping them at -20C? Normal protein storage is at -80C, unless you're working with an enzyme that loses activity upon freezing, in which case -20C in 50% glycerol is acceptable. You can try adding glycerol to 50% to keep the protein cold but not frozen.