From blackhole from abuse.plus.com Fri Oct 3 07:23:18 2008 From: blackhole from abuse.plus.com (Duncan Clark) Date: Fri Oct 3 11:49:08 2008 Subject: PCR help please References: Message-ID: Historians believe that in newspost on Tue, 30 Sep 2008, Becky Pickin penned the following literary masterpiece: > I have tested every >piece I can think of and have run out of ideas...none of my PCR reactions >have resulted in a band - specific or not. OK. Start with something simpler. Prepare a 2x PCR Master Mix minus primers and template i.e. Buffer, Mg, dNTPs, Enzyme and water. Now find someone with M13 forward and Reverse primers and a.n. pUC based plasmid. Could be pUC18, 19, a TA vector etc. with no insert. Using 25pM of each primer and 10ng of plasmid run 20 cycles of PCR using 94C 3mins 94C 5 secs 55C 5 secs 20cycles 72C 15secs 72C 7mins 13C hold Run 5ul on agarose gel and verify it has amplified. That proves your master mix is working. You should be able to dilute the plasmid a few 10fold dilns and still pick up a band noting that supercoiled ccc plasmid will be more refractory to amplification that linear plasmid. Assuming that works go to human DNA and pick a reliable gene to amplify. The following primers amplify fine - pinched from a Fermentas hotstart patent app. GAT GGG CTC TGA GAC TAT AAA GCC GTA GAG AGC TTC CAC CAG GTG TG 402bp product. Works fine with 55C annealing for 10secs and 72C for 30secs extension. Run 30ng, 3ng and 0.3ng for 35cycles loading 5ul of a 50ul PCR on an agarose gel. You should see a feint band at the 0.3ng (100copy) level and obviously much brighter bands at the higher levels. If your master mix is working and those primers do not work then I would blame your template. You can buy inexpensive human DNA as a test control from Sigma - D7011 Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From novalidaddress from nurfuerspam.de Sat Oct 4 03:21:32 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Sat Oct 4 14:41:39 2008 Subject: Affinity chtomatography - Sepharose vs Sephacryl? References: Message-ID: <4ec714cc-c741-4acb-b354-3260a7c50eed@p59g2000hsd.googlegroups.com> Hi Dima, probably this is because most people (have to/want to) stick to established and traditional methods. Those companies who offer the corresponding kits don't need to change their ingredients as long as the existing ones are selling well. Patent issues should be another reason why not all possible combination researchers may think of are available on the market. And in the time of 'publish or perish' none of those who need to apply the methods dares to develop new ones. Also because it becomes more and more difficult to combine the necessary expertise for such developments in one research lab/setting due to its commonly high specialization. just some thoughts, Wolfgang BTW How do you limit shelf-live of your messages? From itisam.sarangi from gmail.com Sat Oct 4 05:31:35 2008 From: itisam.sarangi from gmail.com (Itisam Sarangi) Date: Sat Oct 4 14:42:19 2008 Subject: Help regarding FLAG IP and detergent in elution Message-ID: <25de2fb70810040331w643ed084u399eb44f8acdc63e@mail.gmail.com> hi all, Currently I facing some problems in FLAG IP experiment. I am using the follwoing lysis buffer glycerol 105, tritonx-100 -.9% NP40-.1% Nacl 150mM, EDTA.2mM,HEPES 20mM, PMSF .5mM and prot inhibitor 1x, at ph7.9 I use this washing buffer 150mM nacl, tris 50mM,pH7.4 I perform this expt in a cloumn I have wash 20 column vol of wash buffer. After that I elute using FLAg peptide. chk the bait protein in silver stain. Wash and concentrate it through centricon 2 times and send for MS. yield is in ug level. But MS is not giving any result becoz of some detergent in elution (according to people who perform MS for us) I have tried different wash buffer and centrifuge after each wash cycle so as to minimise the detergent. and even increase washing more in centricon . Centrifuge after each wash cycle in my opninion will decrease any interaction proteins . But at least I am expecting some MS . but still there is detergent in sample. I have thought of follwoing options : 1) decrease the detrgent to .1% NP40. as some protocols use this 2) use hypotonic lysis buffer to wash 3)use some other method to remove detergent using kit form pierce or norgen or beads from sigma Any help in this matter will be very helpful -- Itisam Sarangi From w.ingram from uq.edu.au Sun Oct 5 03:38:03 2008 From: w.ingram from uq.edu.au (Wendy Ingram) Date: Sun Oct 5 15:59:52 2008 Subject: Inert "carrier" for small numbers of tissue culture cells during centrifugation? Message-ID: <48E87CEB.5010501@uq.edu.au> Does anyone know of an inert substance, home made or commercial, that can be added to mammalian cell cultures when spinning? I'm looking for a "carrier" that won't harm live cells, but provides "bulk" to the cell pellet when it would otherwise be very tiny. The goal is to increase recovery when there are very few cells... Hopefully using something that looks different to cells under the microscope when pellets are eventually resuspended. I guess I'm looking for the live cell equivalent of "pellet paint", which is used as a carrier for DNA work. Does anyone know if there is there something similar out there for tissue culture use? Regards, Wendy -- Wendy Ingram, PhD Senior Research Officer RCH Cancer Research Laboratory Department of Paediatrics and Child Health The University of Queensland Australia From virashkgupta from gmail.com Sun Oct 5 11:19:45 2008 From: virashkgupta from gmail.com (Virash Gupta) Date: Sun Oct 5 15:59:59 2008 Subject: Methods Digest, Vol 41, Issue 2 In-Reply-To: <200810031703.m93H3pV23584@net.bio.net> References: <200810031703.m93H3pV23584@net.bio.net> Message-ID: I understand that you must have tried different combinations and have come to depressing conclusions. I have passed through similar phase once. If DNA template is showing good band on agarose, try following simple change but using any RAPD primer. - for 25 microlitre reaction use 5 microlitre of 1 mM (or 1microlitre of 5mM) dNTPmix and amplify for ~ 30 reactions with annealing temperature of ~ 38 degree C. If amplification is good multiband, your everything is good except for primers (specific / random). The most common factor for non amplification is use of higher conc of dNTPs. Try and tell. On Fri, Oct 3, 2008 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: PCR help please (Duncan Clark) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Fri, 3 Oct 2008 13:23:18 +0100 > From: Duncan Clark > Subject: Re: PCR help please > To: methods@net.bio.net > Message-ID: > Content-Type: text/plain;charset=us-ascii;format=flowed > > Historians believe that in newspost > on Tue, 30 Sep 2008, > Becky Pickin penned the following literary > masterpiece: > > I have tested every > >piece I can think of and have run out of ideas...none of my PCR reactions > >have resulted in a band - specific or not. > > OK. > > Start with something simpler. > > Prepare a 2x PCR Master Mix minus primers and template i.e. Buffer, Mg, > dNTPs, Enzyme and water. > > Now find someone with M13 forward and Reverse primers and a.n. pUC based > plasmid. Could be pUC18, 19, a TA vector etc. with no insert. > > Using 25pM of each primer and 10ng of plasmid run 20 cycles of PCR using > > 94C 3mins > > 94C 5 secs > 55C 5 secs 20cycles > 72C 15secs > > 72C 7mins > 13C hold > > Run 5ul on agarose gel and verify it has amplified. That proves your > master mix is working. You should be able to dilute the plasmid a few > 10fold dilns and still pick up a band noting that supercoiled ccc > plasmid will be more refractory to amplification that linear plasmid. > > Assuming that works go to human DNA and pick a reliable gene to amplify. > The following primers amplify fine - pinched from a Fermentas hotstart > patent app. > > GAT GGG CTC TGA GAC TAT AAA GCC > GTA GAG AGC TTC CAC CAG GTG TG > 402bp product. > > Works fine with 55C annealing for 10secs and 72C for 30secs extension. > > Run 30ng, 3ng and 0.3ng for 35cycles loading 5ul of a 50ul PCR on an > agarose gel. You should see a feint band at the 0.3ng (100copy) level > and obviously much brighter bands at the higher levels. > > If your master mix is working and those primers do not work then I would > blame your template. You can buy inexpensive human DNA as a test control > from Sigma - D7011 > > Duncan > -- > I love deadlines. I especially like the whooshing noise they make as > they go flying by. > > Duncan Clark > GeneSys Ltd. > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 41, Issue 2 > ************************************** > -- Dr V K Gupta Sr Microbiologist (Molecular Biology) Insect Molecular Biology Lab Department of Entomology Punjab Agricultural University Ludhiana (Pb)-141004- India M: 09815963210 From novalidaddress from nurfuerspam.de Sun Oct 5 16:23:48 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Sun Oct 5 18:54:49 2008 Subject: Help regarding FLAG IP and detergent in elution References: Message-ID: Maybe you can change the lysis method to something mechanical like potter or high speed shearing (rotor-stator device): When your protein of interest is not membrane bound or transmembrane, detergent should not be a prerequisite to solubilize your protein, as it is already floating in the cytoplasm, so the detergent just would disrupt the membrane and set it free. Wo From nick_theodorakis from hotmail.com Sun Oct 5 19:26:21 2008 From: nick_theodorakis from hotmail.com (Nick Theodorakis) Date: Mon Oct 6 13:57:10 2008 Subject: Inert "carrier" for small numbers of tissue culture cells during centrifugation? In-Reply-To: References: Message-ID: Wendy Ingram wrote: > Does anyone know of an inert substance, home made or commercial, that > can be added to mammalian cell cultures when spinning? I'm looking for a > "carrier" that won't harm live cells, but provides "bulk" to the cell > pellet when it would otherwise be very tiny. The goal is to increase > recovery when there are very few cells... Hopefully using something that > looks different to cells under the microscope when pellets are > eventually resuspended. I guess I'm looking for the live cell equivalent > of "pellet paint", which is used as a carrier for DNA work. Does anyone > know if there is there something similar out there for tissue culture use? > > Regards, > Wendy > As a wild guess, I would look into colored microspheres. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From quangtranho from yahoo.com Sun Oct 5 23:30:44 2008 From: quangtranho from yahoo.com (TRAN HO QUANG) Date: Mon Oct 6 13:57:16 2008 Subject: Helping to isolate DNA that very viscosity Message-ID: <275545.97825.qm@web30405.mail.mud.yahoo.com> Hello, We have isolated DNA from leaves of native species by the CTAB method but we are facing problems: the solution at early and late steps are very viscosity and green colors. Measuring DNA concentration on NanoDrop is nothing. Does anyone has experiences on this issue?. I would like to appreciated to get right comments. Digifellow. From jongh711 from planet.nl Mon Oct 6 05:14:19 2008 From: jongh711 from planet.nl (GysdeJongh) Date: Mon Oct 6 13:57:26 2008 Subject: Inert "carrier" for small numbers of tissue culture cells duringcentrifugation? References: Message-ID: <48e9e4f7$0$27201$ba620dc5@text.nova.planet.nl> "Wendy Ingram" wrote in message news:mailman.256.1223240385.29717.methods@net.bio.net... > Does anyone know of an inert substance, home made or commercial, that > can be added to mammalian cell cultures when spinning? I'm looking for a > "carrier" that won't harm live cells, but provides "bulk" to the cell > pellet when it would otherwise be very tiny. The goal is to increase > recovery when there are very few cells... Hopefully using something that > looks different to cells under the microscope when pellets are > eventually resuspended. I guess I'm looking for the live cell equivalent > of "pellet paint", which is used as a carrier for DNA work. Does anyone > know if there is there something similar out there for tissue culture use? Hi Wendy Ingram, surprising question.I just wonder why do you want to do this ?? I did a lot of cell culture in my career and I frequently purified low numbers of cells.From FAC's experiments for instance. Here are a few tricks : I found that the biggest problem is the plastic tube and just the mechanical mixing.Cells tend to stick to plastic irreversibly and are whirled up easily because they are much heavier than water. 1) Use conical centrifuge tubes, this will prevent un-intended re-mixing 2) Cool all media to 4 degrees before you start.The sticking of cells is temp dependant 3) Rinse _every thing_ you are going to use with a cold solution of about 1% bovine serum albumine or, even better, fetal calf serum.Also plastic pipets 4) The weight of the cells is much higher than water; they are very fragile after all the previuous maniulations.Use the lowest centrifugal force that works for you.Or increase the time first and not the rpm.I used 200 g for 10 minutes. 5) Never aspirate _all_ of the supernatant.If you want thorough washing than increase the number of washing steps. 6) Never use a vortex.It will cause clumbs.Use a disposable (pre-rinsed !!!) plastic pipet and position it _above_ and not just "_in_ " your (supposed) pellet.Then very slowly move the supernatant up- and down. 7) If you need the precise volume do this by weighing the tube with- and without your cell suspension.Don't remove all the supernatant just for this pupose. 8) A Burker chamber with 0.5 microliter of the cell suspension can be used to do a quick check on the quality of the cells under a phase contrast microscope at a 25 magnification. hth Gys From ijpinto from ibmc.up.pt Mon Oct 6 14:12:46 2008 From: ijpinto from ibmc.up.pt (ijpinto@ibmc.up.pt) Date: Tue Oct 7 10:51:45 2008 Subject: T cell activation Message-ID: Dear all, I'm having a problem in inducing proliferation of CD4+ and CD8+ cells in vitro. I've coated 96 well plates with 10 micrograms/ml anti-CD3 (OKT3) and added 2 micrograms/ml soluble anti-CD28. After 4 days of incubation I still don't get any proliferation. These same conditions worked well for PBMCs. My specific questions are: 1- Do I have to perform any special treatment of the plates before adding the anti-CD3 antibody? I'm just adding the antibody, incubating 3 hours at 37?C, washing with HBSS and adding the cells right afterwards. However, I saw some studies where they add goat anti-mouse IgG prior to the OKT3. Does this makes an essential difference? 2- Do I have to supplement with IL-2, in addition to anti-CD3 and anti-CD28, to get T cell proliferation? Your help is welcomed. Thanks a lot! Jorge Jorge P Pinto, PhD Iron Genes and Immune System Cell Culture and Genotyping (Head) Institute for Molecular and Cell Biology Rua do Campo Alegre, 823 4150-180 Porto Portugal Phone: +351-226074956 email: ijpinto@ibmc.up.pt From R.Jayakumar from roswellpark.org Tue Oct 7 11:08:40 2008 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Tue Oct 7 13:10:36 2008 Subject: T cell activation In-Reply-To: References: Message-ID: <97101976F8A044468CA74FE11883B90E173E7B5F@VISTA.roswellpark.org> Wow.. Precoating the plates is good but really not necessary for inducing T-cell proliferation. Just CD3 coated plates alone should be able to induce T-cell proliferation to a lower level. You can add both anti-CD3 and anti-CD28 as soluble. Add the required amount of T-cells in each well (50,000-100,000 cells or so), add anti-CD3 (OKT3 clone)and anti-CD28 (clone 28.2 or Mab 9.3) and it should proliferate. What kind of Mab clones are you working with here (regarding anti-CD3 and anti-CD28)? No need to supplement with IL-2, since IL-2 induction is what causes T-cell proliferation after you ligate TCR and CD28. You could also try using a positive control with PMA and calcium ionophore, which will definitely induce T-cell proflieration if your T-cells are viable. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of ijpinto@ibmc.up.pt Sent: Monday, October 06, 2008 3:13 PM To: methods@magpie.bio.indiana.edu Subject: T cell activation Dear all, I'm having a problem in inducing proliferation of CD4+ and CD8+ cells in vitro. I've coated 96 well plates with 10 micrograms/ml anti-CD3 (OKT3) and added 2 micrograms/ml soluble anti-CD28. After 4 days of incubation I still don't get any proliferation. These same conditions worked well for PBMCs. My specific questions are: 1- Do I have to perform any special treatment of the plates before adding the anti-CD3 antibody? I'm just adding the antibody, incubating 3 hours at 37?C, washing with HBSS and adding the cells right afterwards. However, I saw some studies where they add goat anti-mouse IgG prior to the OKT3. Does this makes an essential difference? 2- Do I have to supplement with IL-2, in addition to anti-CD3 and anti-CD28, to get T cell proliferation? Your help is welcomed. Thanks a lot! Jorge Jorge P Pinto, PhD Iron Genes and Immune System Cell Culture and Genotyping (Head) Institute for Molecular and Cell Biology Rua do Campo Alegre, 823 4150-180 Porto Portugal Phone: +351-226074956 email: ijpinto@ibmc.up.pt _______________________________________________ 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 ebrahimsotoudeh from gmail.com Wed Oct 8 02:50:22 2008 From: ebrahimsotoudeh from gmail.com (Ebrahim Sotoudeh) Date: Wed Oct 8 11:40:13 2008 Subject: method for measurement of alkaline phosphatase ... Message-ID: <6ba5fcfa0810080050q3ef6304eka9d872a871e4441d@mail.gmail.com> Hello I'm an MSc student of fisheries engineering at Tarbiat Modares university. The title of my thesis is "Effect of Dietary Soybean Lecithin on Growth, Fatty Acid Profile and Enzyme Activity of Caspian Salmon (salmo trutta caspius Kessler 1877) fry". I want to assay alkaline phosphatase activity. and I need method for * measurement* of *alkaline phosphatase* activity . could you help me best regard Ebrahim Sotoudeh From ivanoov from gmail.com Wed Oct 8 03:14:37 2008 From: ivanoov from gmail.com (chovek69) Date: Wed Oct 8 11:40:20 2008 Subject: Helping to isolate DNA that very viscosity References: Message-ID: The simple solution would be to try with less starting material. Increase also incubation time with Prot K or its amount. Undegisted proteins could contribute to viscosity. The greenish color can be removed (or at least lessen) by doing one extraction with only phenol before the phenol/ chloroform step. The precipitation should be performed by using less centrifugation speed (say 10 000 rpm instead of 13k rpm). Washing with 70% ethanol however is on max speed. Hope this helps TRAN HO QUANG wrote: > Hello, > > We have isolated DNA from leaves of native species by the CTAB method but we are facing problems: the solution at early and late steps are very viscosity and green colors. Measuring DNA concentration on NanoDrop is nothing. > > > Does anyone has experiences on this issue?. I would like to appreciated to get right comments. > > Digifellow. From shifalich from gmail.com Wed Oct 8 23:41:05 2008 From: shifalich from gmail.com (shifalich@gmail.com) Date: Thu Oct 9 10:52:03 2008 Subject: PCR help please References: Message-ID: <617d65b6-bdeb-4b41-a2a3-387d12069408@p31g2000prf.googlegroups.com> On Sep 30, 11:32?pm, "Becky Pickin" wrote: > Hello all, > > I am back again this time asking for help with some problem PCR. ?I will do > my best to explain what I have done. ?If you have any suggestions or > tips/tricks I am quite willing to give them a try. ?I have tested every > piece I can think of and have run out of ideas...none of my PCR reactions > have resulted in a band - specific or not. ?All products should be between > 200bp and 1200bp. ?(negative controls are always blank - no streak, smear, > band..nothing - very clean) > > Let me start with what I'm doing: > > PCR on HeLa cell genomic DNA both EcoRI digested and not. ?The initial > template used was <1 yr old HeLa genomic DNA isolated in our lab by another > member (excellent lab hands) and digested with EcoRI. ?He has used this > template in PCR and for probe design within the past 6 months. > Concentration has been re-checked using a BioRad Spec measuring A260/280. > In addition, dilutions (down to the concentration used in PCR reactions) of > the DNA were run on a gel and resulted in the predicted smear (b/c it was > EcoRI digested). Alternative templates were freshly isolated (within the > past 2 weeks) HeLa genomic DNA not EcoRI digested and a purified plasmid > DNA. ?All templates have been diluted to ~20ng/ul and 1ul added to the PCR > reaction. ?Templates (except for the undigested genomic) have been used > successfully by other lab members. > > I designed 7 pairs of primers using the Primer 3 program and double checking > primer dimers, etc. using the oligo calculator at Northwestern. ?I have > experience designing primers and had another lab member (faculty) double > check them before I ordered the primers. ?Primers are designed to have a Tm > of 55-65oC depending on how you calculate (ie salts, etc.) ?The target Tm > was 60oC. ?Chosen pairs were not anticipated to have any hairpins, primer > dimers, etc (I had enough flexibility to select against any that were > predicted to be problematic in that regards). ?These are the "experimental" > primers. ?All primers are reconstituted in TE at 100uM and then diluted to > 10uM in autoclaved ddH2O for a working stock. ?These were ordered and > reconstituted within the past 2 weeks. > > Positive control primers are also at 10uM working stocks and have been shown > to give a band with the EcoRI digested HeLa genomic DNA within the week by > another lab member (as part of the trouble shooting process) in QPCR. > Dissociation curves show a specific product using Sybr Green chemistry. > These primers have also been used successfully by other lab members (using > more stringent reaction conditions, annealing temp of 60oC) and was why they > were selected as a positive control for these experiments. ?I have been > unsuccessful at obtaining a product with these primers using any of the > three templates listed above. > > My reaction chemistry is as follows: > ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? * Conc in > reaction* ? ? *Volume used (ul)/rxn* > 10x PCR Buffer (-MgCl2) from Invitrogen > 1x ? ? ? ? ? ? ? ? ? ? ? ? ? ?5 > 50mM MgCl2 > 1.5mM ? ? ? ? ? ? ? ? ? ? 1.5 > 5u/ul Taq Polymerase > 1u ? ? ? ? ? ? ? ? ? ? ? ? ? ?0.5 > (these are all part of Invitrogen Platinum Taq Polymerase "kit" Cat > #10966-026 and are fresh and have been used in a PCR by another lab member > and resulted in a positive band) > Autoclaved ddH2O > - ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?40 > 10mM dNTP mix ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0.2mM > @ ? ? ? ? ? ? ? ? 1 > Primer 1 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0.2uM > @ ? ? ? ? ? ? ? ? ?0.5 > Primer 2 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0.2uM > @ ? ? ? ? ? ? ? ? ?0.5 > Template (diluted to 20ng/ul) > 20ng ? ? ? ? ? ? ? ? ? * ? ? 1 ? ? ? * > > 50ul per reaction > > dNTPs are from Roche diagnostics (#11277049001) shipped as 100mM lithium > salt solutions at pH7 (10umol in 100ul). ?I mixed the 4 dNTPs together as > such: 20ul of @dNTP + 120ul ddH2O, made within the past 2 weeks and stored > at -20oC. > > Reactions are set up with a premix adding water followed by buffer first. > Some reactions had template added separately, some reactions had primers > added separately. > > Reaction Conditions tried: > Try 1: > Step 1 94oC ?2min > ? ? ? ? ? 57oC ? 1min ? ? ?1 cycle > ? ? ? ? ? 72oC ? 2 min > > Step 2 ?94oC ?1min > ? ? ? ? ? ?57oC ? 1min ?28 cycles > ? ? ? ? ? ?72oC ? 2min > > Step 3 ?94oC ?1min > ? ? ? ? ? 57oC ? 1min ? ? ? 1 cycle > ? ? ? ? ? ?72oC ? 10min > > Step 4 ?Hold at 4oC > > Try 2: > Same as Try 1 with annealing temperature reduced to 55oC, total cycles > increased to 40 > > Try 3: > Step 1 94oC ?2min > ? ? ? ? ? 55oC ? 1min ? ?1 cycle > ? ? ? ? ? 72oC ? 2 min > > Step 2 ?94oC ?30 sec > ? ? ? ? ? ?55oC ? 30 sec ? 38 cycles > ? ? ? ? ? ?72oC ? 2min > > Step 3 ?94oC ?30 sec > ? ? ? ? ? ?55oC ? 30 sec ? ?1 cycle > ? ? ? ? ? ?72oC ? 10min > > Step 4 ?Hold at 4oC > > 2 different thermocyclers have been tried. ?Both have a heated lid and thus > no oil was applied to the reactions. ?PCR "products" have loading dye added > and then are applied to between 1% and 1.5% agarose gels. ?The gels are run > at 100-120 volts until the dye front migrates ~2/3 of the way down the gel. > Gels are then stained with EtBr and photographed on UV light box. ?Markers > used are beautiful. > > Help please! > > Thank you, > R Pickin, PhD Dear pickin! Can you please tell, how you are making mix of dNTP again? Shifali From taskan4 from gmail.com Thu Oct 9 04:30:56 2008 From: taskan4 from gmail.com (TR) Date: Thu Oct 9 10:52:09 2008 Subject: Will SNAP i.d. increade my happiness? Message-ID: <70272d730810090230x61032af3tcdb5288cc41083c0@mail.gmail.com> Dear western-blot experts, A sales rep. from Millipore just left the lab and almost convinced me to buy what he calls "a revolution in your western-blot experience": a simple device (SNAP i.d.) that makes all incubations shorter by forcing all solutions "through" the membrane (using vacuum). http://www.millipore.com/immunodetection/id3/snap_id It certainly looks like a much simpler, quicker and cleaner way to do all incubation for the westerns, but I would like to hear from people who have actually used the mechine. The question is: will the 850 euros investment increase our happiness? All the best CG From jg374 from mole.bioc.cam.ac.uk Thu Oct 9 05:50:11 2008 From: jg374 from mole.bioc.cam.ac.uk (James Giddings) Date: Thu Oct 9 10:52:34 2008 Subject: "tissue culture treated"? Message-ID: What does "tissue culture treated" actually mean in relation to plasticware? Is it coated with something? Why do cells adhere better than to standard polystyrene plates? From kgilbride from shire.com Thu Oct 9 11:50:54 2008 From: kgilbride from shire.com (Gilbride, Kevin) Date: Thu Oct 9 13:16:41 2008 Subject: "tissue culture treated"? In-Reply-To: References: Message-ID: <0F2CFCE6FC067A42813DFA1F9BB0697C01A7E566@SHCHBEX03.corp.shire.com> I believe the TC plastic ware carries a small static charge that helps the cells adhere better. This is the only difference I'm aware of between the two. I've done experiments that required adherent cells to remain in suspension and have also dealt with this issue. Kevin -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of James Giddings Sent: Thursday, October 09, 2008 6:50 AM To: methods@magpie.bio.indiana.edu Subject: "tissue culture treated"? What does "tissue culture treated" actually mean in relation to plasticware? Is it coated with something? Why do cells adhere better than to standard polystyrene plates? _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods ______________________________________________________________ This message has been scanned for all viruses by BTnet VirusScreen. The service is delivered in partnership with MessageLabs. This service does not scan any password protected or encrypted attachments. If you are interested in finding out more about the service, please visit our website at http://www.btignite.com/internetservices/btnet/products_virusscreen.htm ============================================================== Please consider the environment before printing this e-mail This email and any files transmitted with it are confidential and may be legally privileged and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient please note that any disclosure, distribution, or copying of this email is strictly prohibited and may be unlawful. If received in error, please delete this email and any attachments and confirm this to the sender. www.shire.com From aawara from pontiff-playground.org Thu Oct 9 20:16:29 2008 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Thu Oct 9 23:32:52 2008 Subject: Will SNAP i.d. increade my happiness? References: Message-ID: In , TR wrote: > It certainly looks like a much simpler, quicker and cleaner way to do > all incubation for the westerns, but I would like to hear from people > who have actually used the mechine. The question is: will the 850 > euros investment increase our happiness? We've been testing it for the last 3 weeks. So far, it has proven to not work as well doing our incubations in little plastic bags. The background is very low (we use 0.125% non-fat milk with the SNAP i.d., and 5% non-fat milk for blots done the old fashioned way), however, the sensitivity is much better the old fashioned way (about 10 - 50 times more sensitive). AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From anirbn from gmail.com Thu Oct 9 22:54:33 2008 From: anirbn from gmail.com (ab) Date: Thu Oct 9 23:33:01 2008 Subject: Most efficient PCR strategy for generating fusion (chimeric) proteins ? Message-ID: <38bf6e5f-761d-438c-a571-a6ab80af4ce1@j22g2000hsf.googlegroups.com> Dear all, I have to generate several chimeric proteins where I will have to insert about 50 aa residues of one protein in the middle of another. I was searching through the archives and found the Emerman, Deminie protocol ( J.Virol , July 1993 p.6499-6506). Basically one has to do four PCR reactions followed by a restriction digest and ligation. Is this still the most efficient way to generate chimeric proteins where the insertion size is relatively big ? Any help will be really appreciated. Best regards, AB. From anirbn from gmail.com Thu Oct 9 23:02:03 2008 From: anirbn from gmail.com (ab) Date: Thu Oct 9 23:33:10 2008 Subject: How reliable is ligation independent cloning ? Message-ID: Dear all, I have to generate a large number of constructs for different genes and am considering ligation independent cloning. However, someone told me today that ligation independent cloning is not that reliable and can give rise to insertions/mutations . Does that agree with your experience ? Will you please share ? What will be your advice ? Thanks a ton in advance. AB. From nick.theodorakis from gmail.com Fri Oct 10 10:24:36 2008 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Fri Oct 10 15:20:20 2008 Subject: George Palade dies Message-ID: <4169977a-8eb6-4427-84b6-8b49323545ae@o40g2000prn.googlegroups.com> NY Times article: LOS ANGELES =97 George E. Palade, whose discoveries about the intricate inner workings of cells helped give birth to the field of modern cell biology and earned him a Nobel Prize, died Tuesday at his home in Del Mar, Calif., at 95 . . . . ----- Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From arnec from bio.umass.edu Fri Oct 10 13:47:16 2008 From: arnec from bio.umass.edu (Arne K Christensen) Date: Fri Oct 10 15:20:32 2008 Subject: commercially available in situ probes Message-ID: <48EFA334.1070102@bio.umass.edu> I am looking for a commercial source of in situ probes. I am wondering if anybody has ordered DIG-labeled in situ probes, and used them successfully. Thank you, Arne -- __ Arne K Christensen Postdoctoral Research Associate University of Massachusetts, Amherst USGS Conte Anadromous Fish Research Center One Migratory Way, PO Box 796 Turners Falls, MA 01376 Email: arnec@bio.umass.edu Phone: (413) 863-3827 Fax: (413) 863-9810 URL: www.biobog.com From lvkaiyang from hotmail.com Sat Oct 11 09:42:59 2008 From: lvkaiyang from hotmail.com (kaiyang) Date: Sat Oct 11 12:17:51 2008 Subject: iNOS Western Problems Message-ID: Hi! Do you have a iNOS western protocol? I'm having problems western blotting for iNOS too! Have you succeeded ? Kai yang LV ChianFrom zineldeen from gmail.com Sun Oct 12 10:24:16 2008 From: zineldeen from gmail.com (doaa zineldeen) Date: Sun Oct 12 12:12:38 2008 Subject: Methods Digest, Vol 41, Issue 10 In-Reply-To: <200810111704.m9BH4TV09592@net.bio.net> References: <200810111704.m9BH4TV09592@net.bio.net> Message-ID: <440923e10810120824n36b7e841n6f4e2add747fb9ca@mail.gmail.com> Dear all I am going to perform PI/FITC double staining of U2OS cells by FACScan it is my first time to use this cell line for FACS I am wondering is the set up of these cells similar to that of 3T3 cells or not any suggestion is appreciated doaa From cassiha from gmail.com Tue Oct 14 07:35:35 2008 From: cassiha from gmail.com (hilary cassidy) Date: Tue Oct 14 11:25:53 2008 Subject: Searching for small RNAs Message-ID: <95ef7c150810140535k4afef0f3u186493193446e04f@mail.gmail.com> Hey everyone, I was working to clone small RNAs from HPV for my masters and have cloned these small RNAs and sequenced the results. To finish this work I needed to scan the sequences for the presence of human miRNAs, unique miRNAs, mRNAs, viral genomic loci etc. I am stuck on how to search for the rRNAs and tRNAs and my supervisors don't know the answers. I was wondering if someone could guide me to which website/database to use to search a sequence for the presence of rRNA and tRNA? Any help would be greatly appreciated. Thanks in advance, Regards, Hilary From engelbert_buxbaum from hotmail.com Tue Oct 14 14:48:47 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Oct 14 15:56:31 2008 Subject: Affinity chtomatography - Sepharose vs Sephacryl? References: <4ec714cc-c741-4acb-b354-3260a7c50eed@p59g2000hsd.googlegroups.com> Message-ID: Am 04.10.2008, 20:53 Uhr, schrieb DK : > In article > <4ec714cc-c741-4acb-b354-3260a7c50eed@p59g2000hsd.googlegroups.com>, WS > wrote: >> >> probably this is because most people (have to/want to) stick to >> established and traditional methods. Those companies who offer the >> corresponding kits don't need to change their ingredients as long as >> the existing ones are selling well. > > Well, I asked for a *good* reason :-) That's not quite it. Basically, I > am > trying to understand why Pharmacia itself never sold any form of > activated or ligand-coupled Sephacryl and is selling a dozen of > various activated and affinity sorbents based on Sepharose. Part of the reason may be that the old Pharmacia company no longer exists, it was bought first by Amersham and later by GE. It is quite possible that the expertise in separation technology once concentrated at Pharmacia has dispersed. The fact that the monographs on various separation techniques that Pharmacia used to offer for free are no longer maintained or even available (and it would be so cheap to put a pdf on their web-site) strongly points in that direction, as does the fact that we no longer hear about new techniques or matrices developed there. At least the Hoefer electrophoresis line is now rescued. Sic transit gloria mundi :-( From rmorissette from jhu.edu Tue Oct 14 13:12:44 2008 From: rmorissette from jhu.edu (Rachel Morissette) Date: Tue Oct 14 15:56:41 2008 Subject: anti-CRD antibody Message-ID: Hi everyone, I am trying to locate a commerical (or private if anyone has any available) source of the anti-CRD (cross-reacting determinant) antibody for use in detecting the GPI anchor. The one place we found (Prozyme) no longer makes it and so far it seems that raising it in rabbits and purifying it is the only means of obtaining this antibody (which is not an option at this point due to time constraints). Can anyone provide any help for our dilemma? Thanks, Rachel Rachel Morissette, Ph.D. Johns Hopkins University, School of Medicine Department of Biophysics and Biophysical Chemistry 725 N. Wolfe Street, Hunterian 709 Baltimore, MD 21205 410-955-3077 From engelbert_buxbaum from hotmail.com Tue Oct 14 15:06:21 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Oct 14 15:56:55 2008 Subject: method for measurement of alkaline phosphatase ... References: Message-ID: Am 08.10.2008, 03:50 Uhr, schrieb Ebrahim Sotoudeh : > I want to assay alkaline phosphatase activity. and I need method for * > measurement* of *alkaline phosphatase* activity . The cheapest way to do this is to follow the hydrolysis of para-nitrophenylphosphate (pNPP) (colourless) to nitrophenol (yellow in alkaline solutions) photometrically at 405 nm. Acid phosphatase is measured in citrate buffer pH 5.6, alkaline phosphatase at pH 8-11 (you need to find out the optimum for your enzyme, check BRENDA for published data). If sample size are limited you can use methylumbelliferyl phosphate instead, which gives a fluorescent product. From nick.theodorakis from gmail.com Wed Oct 15 00:02:36 2008 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Wed Oct 15 09:18:02 2008 Subject: Affinity chtomatography - Sepharose vs Sephacryl? References: <4ec714cc-c741-4acb-b354-3260a7c50eed@p59g2000hsd.googlegroups.com> Message-ID: <36283c4c-b29c-4297-a977-b38e2c40d6a6@f40g2000pri.googlegroups.com> On Oct 14, 3:48 pm, "Dr Engelbert Buxbaum" wrote: ... > > Part of the reason may be that the old Pharmacia company no longer exists, > it was bought first by Amersham and later by GE. It is quite possible that > the expertise in separation technology once concentrated at Pharmacia has > dispersed. The fact that the monographs on various separation techniques > that Pharmacia used to offer for free are no longer maintained or even > available (and it would be so cheap to put a pdf on their web-site) > strongly points in that direction, as does the fact that we no longer hear > about new techniques or matrices developed there. I actually learned quite a bit from those booklets. Everything I needed to know about gel filtration I learned from Pharmacia ;-) Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From legatek from hotmail.com Thu Oct 16 00:36:45 2008 From: legatek from hotmail.com (Kyle Legate) Date: Thu Oct 16 12:16:06 2008 Subject: Will SNAP i.d. increade my happiness? In-Reply-To: References: Message-ID: <6lo27cFd8io4U1@mid.individual.net> TR wrote: > Dear western-blot experts, > > A sales rep. from Millipore just left the lab and almost convinced me > to buy what he calls "a revolution in your western-blot experience": a > simple device (SNAP i.d.) that makes all incubations shorter by > forcing all solutions "through" the membrane (using vacuum). > http://www.millipore.com/immunodetection/id3/snap_id > > It certainly looks like a much simpler, quicker and cleaner way to do > all incubation for the westerns, but I would like to hear from people > who have actually used the mechine. The question is: will the 850 > euros investment increase our happiness? > > All the best > CG > You will be happy until you have to order new cassettes. From smhowell from usc.edu Fri Oct 17 20:28:06 2008 From: smhowell from usc.edu (Shannon Howell) Date: Fri Oct 17 21:29:43 2008 Subject: LNA Message-ID: Hi, I saw your thread......shown below....and was wondering if you got an answer. I am trying to transfect LNA into 293 cells as well. Thanks! Shannon Hi everyone! I've got a problem, maybe one of can help me. I want to transfect Locked nucleic acids (LNAs) into 293T cells. HAs anyone of you expereince with LNA transfection and whch transfection reagent do you use? Most publication use Lipofectamin. At the moment im using Metafectene (www.biontex.com). At the moment it seems that knock down somehow works, but I can't find out easily how efficient LNAs get transfected- so I'd need some transfection reagent to comapre it to! Thanks for your answers! Andi From Andre.Hamel from gov.mb.ca Wed Oct 22 11:14:11 2008 From: Andre.Hamel from gov.mb.ca (Hamel, Andre (MAFRI)) Date: Wed Oct 22 14:29:32 2008 Subject: Detection enhancer in Ambion AgPath 1-step real time RTPCR kit Message-ID: <9A52FA4A0436FC489251BAB5249DAADC03414780@OC1EX01.ME.MBGOV.CA> Hi, I have some questions/concerns about Ambion's AgPath 1-step real time RTPCR kit & their "detection enhancer" ("DE"). 1 - Does anyone have experience with the performance of comparing with & without DE? 2 - Has anyone obtained any written information from folks at ABI and/or Ambion regarding NOT using the DE with their kit? 3 - Does anyone have any idea as to what the DE consists of? - for example is it simply betaine? Thanks, Andre Hamel, Scientist (MSc) Virology, Veterinary Diagnostic Services Manitoba Agriculture, Food and Rural Initiatives (MAFRI) 545 University Crescent, Winnipeg, Manitoba, Canada R3T 5S6 Ph:(204)945-7651; Fax:(204)945-8062; Andre.Hamel@gov.mb.ca From amitsk84 from yahoo.co.in Thu Oct 23 01:40:25 2008 From: amitsk84 from yahoo.co.in (Amit) Date: Thu Oct 23 09:14:39 2008 Subject: Protein elution from PVDF membrane Message-ID: <49221.24462.qm@web8703.mail.in.yahoo.com> Dear Sir/ Madam,, I would like to state that i am a research assistant here in India, at post graduate institute for medical sciences and research, Chandigarh. Presently we are working on some protein profiling and some work with reverse genetics. In this process we are in need of some protocols or methods that can work with binding of some antibodies (in serum form) on either nitrocellulose membrane or PVDF membrane. After binding of the antibodies we are looking for the elution of proteins of interest from the membrane. in this process we are looking for some standardized protocol or methods. I got this mail address a link from the website www.bio.net.com I will very grateful to you if you can suggest some methods or mail some protocol for this. thanking you. Best regards, Amit Khatri Research Assistant PGIMER, Chandigarh India Connect with friends all over the world. Get Yahoo! India Messenger at http://in.messenger.yahoo.com/?wm=n/ From marker from rhrk.uni-kl.de Thu Oct 23 09:53:36 2008 From: marker from rhrk.uni-kl.de (Simone Marker) Date: Thu Oct 23 13:03:20 2008 Subject: phosphatase removal in RNA samples Message-ID: Hi, I am looking for chemical properties of short RNA species (siRNA) and I want to know wheather they are 5' phosphorylated (e.g. mono-or triphosphate). I want to compare their migration behaviour in a denaturing polyacrylamide gel (7M urea), which is also influenced by the number of phosphates. I also need some RNA samples that are completely dephosphorylated, to compare the migration. So I would use calf intestinal alkaline phosphatase (better than SAP with RNA), but I do not want to remove the phosphatase by phenol-chlorophorm extraction (to much loss of RNA, not quatitative). Do you think that removal is necessary? I just want to load the treated samples on the gel (near the untreated ones) and see their migration behaviour (in northern). Does active phosphatase influence the migration of the RNA in denaturing gels? (since CIP is not heat-inactivatable). Thank you for your help, Simone From TCruz from ibmc.up.pt Thu Oct 23 11:37:42 2008 From: TCruz from ibmc.up.pt (TCruz@ibmc.up.pt) Date: Thu Oct 23 13:03:28 2008 Subject: Membrane proteins Message-ID: Dear all, I work with membrane proteins of Leishmania and I have b= een having some problems. First= , does anyone have experience with chimeras? I did a GFP and a c-cmyc fusio= n protein and none of them could be detected by western blot. Both fusions = are C-terminal, though I also did two N-terminal c-myc tagged proteins and = again nothing was detected. = Sec= ond, I tried to produce antibodies against those membrane proteins using re= combinant polypeptide and although it recognizes the recombinant truncated = protein, it does not recognize in parasite total extracts (in SDS-PAGE). Is= this common? = Due to this result= I introduced an episomic copy of the gene in order to over-express the pro= teins in the parasites. Since I could not detect the proteins by western bl= ot, I went back to qPCR trying to understand if there were at least higher = levels of mRNA. Indeed I saw a fold increase of around 70 for one of the pr= oteins and 4 for another one. The other 2 proteins did not show any increas= e in the mRNA levels. So, in conclusion, I wonder if the parasites could be able to eli minate the mRNA so fast that no protein is detected and how come the parasi= tes don't have any mRNA if they carry an episomic copy of the gene in= serted in an expression vector. I know this is a lot, but can you help me? I thank you all in advance, T?nia Cruz Iron Genes and Immune System Institute for Molecular a= nd Cell Biology Rua do Campo Alegre, 823 4150-180 Porto PortugalPhone: +351-226074956 email: [1]tcruz@ibmc.up.pt References 1. 3D"mailto:tcruz@ibmc.up.pt" From novalidaddress from nurfuerspam.de Thu Oct 23 15:05:19 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Thu Oct 23 16:23:12 2008 Subject: phosphatase removal in RNA samples References: Message-ID: <9df7a95f-ae31-43ac-b30d-4d93408c1a50@d70g2000hsc.googlegroups.com> On Oct 23, 4:53?pm, "Simone Marker" wrote: > Hi, > > I am looking for chemical properties of short RNA species (siRNA) and I want > to know wheather they are 5' phosphorylated (e.g. mono-or triphosphate). I Hi Simone, two things you could try: 1) add a tiny little bit of phenol to the sample. This will kill the phosphatease. No need for any extraction. 2) If this should not be an option, you might add 100mM sodium fluoride to the gel buffers to inhibit the phosphatase. Will increase conductivity however and thus heat production during electrophoresis. Best reagrds, Wo, from Bochum > want to compare their migration behaviour in a denaturing polyacrylamide gel > (7M urea), which is also influenced by the number of phosphates. I also need > some RNA samples that are completely dephosphorylated, to compare the > migration. > So I would use calf intestinal alkaline phosphatase (better than SAP with > RNA), but I do not want to remove the phosphatase by phenol-chlorophorm > extraction (to much loss of RNA, not quatitative). Do you think that removal > is necessary? I just want to load the treated samples on the gel (near the > untreated ones) and see their migration behaviour (in northern). Does active > phosphatase influence the migration of the RNA in denaturing gels? (since > CIP is not heat-inactivatable). > > Thank you for your help, > Simone From shenq from purdue.edu Thu Oct 23 15:17:22 2008 From: shenq from purdue.edu (Qingwu Shen) Date: Thu Oct 23 16:23:20 2008 Subject: A question about pET system Message-ID: <1224793042.4900dbd2dedfc@webmail.purdue.edu> Hi everybody, I have a question about pET system. As you know, pET vectors do not have lacZ ¦Á- peptide and are not applicable to blue/white screening. However, why the colonies still become blue on plate when XL1 blue transformed with pET28 basic vector is grown for more than 36 hours? Thank you very much. shen From biocjh from gmail.com Thu Oct 23 21:40:45 2008 From: biocjh from gmail.com (jh) Date: Thu Oct 23 23:13:14 2008 Subject: Protein elution from PVDF membrane In-Reply-To: <49221.24462.qm@web8703.mail.in.yahoo.com> References: <49221.24462.qm@web8703.mail.in.yahoo.com> Message-ID: maybe high salt solution will help. Jianhao 2008/10/23 Amit : > Dear Sir/ Madam,, > > I would like to state that i am a research assistant here in India, at post graduate institute for medical sciences and research, Chandigarh. Presently we are working on some protein profiling and some work with reverse genetics. In this process we are in need of some protocols or methods that can work with binding of some antibodies (in serum form) on either nitrocellulose membrane or PVDF membrane. After binding of the antibodies we are looking for the elution of proteins of interest from the membrane. in this process we are looking for some standardized protocol or methods. > I got this mail address a link from the website www.bio.net.com > > I will very grateful to you if you can suggest some methods or mail some protocol for this. > > thanking you. > > Best regards, > Amit Khatri > Research Assistant > PGIMER, Chandigarh > India > > > Connect with friends all over the world. Get Yahoo! India Messenger at http://in.messenger.yahoo.com/?wm=n/ > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From sadresm2002 from yahoo.com Thu Oct 23 11:39:38 2008 From: sadresm2002 from yahoo.com (Esmaeil Sadroddiny) Date: Thu Oct 23 23:13:20 2008 Subject: ppm in mass spectrometry Message-ID: <153986.46203.qm@web32601.mail.mud.yahoo.com> Can somebody explain simple definition from ppm in mass spectrometry Regards Esmaeil From ivanoov from gmail.com Fri Oct 24 05:04:05 2008 From: ivanoov from gmail.com (chovek69) Date: Fri Oct 24 11:09:36 2008 Subject: RT-PCR question Message-ID: <128edaa6-4367-41e9-ba53-9daa484100e5@e17g2000hsg.googlegroups.com> Dear experts, I am relatively new to the RT-PCR world and just would like to hear some suggestions about some basic practices: Usually I come up with the RNAs in the evening so what is better: to run RT-PCR with hexamers overnight (cDNA stay at room temp whole night) or to freeze the RNA at -80C and do the RT-PCR on the next day ? I am to run PCRs for viral detection (low-copy number) in ticks. Thanks From nobody from nospam.not Fri Oct 24 06:33:01 2008 From: nobody from nospam.not (Han) Date: Fri Oct 24 11:09:42 2008 Subject: ppm in mass spectrometry References: Message-ID: Esmaeil Sadroddiny wrote in news:mailman.484.1224821707.29717.methods@net.bio.net: > Can somebody explain simple definition from ppm in mass spectrometry > > Regards > Esmaeil > p parts p per m million Usually weight/weight, AFAIK -- Best regards Han email address is invalid From bcgau from artsci.wustl.edu Fri Oct 24 10:07:21 2008 From: bcgau from artsci.wustl.edu (Brian Gau) Date: Fri Oct 24 11:09:49 2008 Subject: ppm in mass spectrometry In-Reply-To: <153986.46203.qm@web32601.mail.mud.yahoo.com> References: <153986.46203.qm@web32601.mail.mud.yahoo.com> Message-ID: <000301c935ea$38df0890$aa9d19b0$@wustl.edu> Hi Parts-per-million: The exact mass/charge of an ion is calculated from the mono-isotopic mass, not the average, for each element in its elemental composition. An electron mass (or several) must be added or taken away to get the exact ion mass/charge (and divide by the charge if doubly, triply, etc. charged). The ppm difference between an observed ion mass/charge and exact ion mass/charge is simply: (observed - exact)/exact X 1000000 In general a |ppm| difference smaller than 5 is quite good agreement, though the best Fourier transform ion cyclotron resonance and Orbitrap spectrometers deliver sub ppm accuracy. This all supposes you know what you're looking at. If you're looking at an unknown, then you take confidence in its measured mass when the calibrated spectrometer verifies the calibrant mass/charge values to within 5 or better ppm. Good Friday, Brian -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Esmaeil Sadroddiny Sent: Thursday, October 23, 2008 11:40 AM To: methods@net.bio.net Subject: ppm in mass spectrometry Can somebody explain simple definition from ppm in mass spectrometry Regards Esmaeil _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From bmacgreg from unc.edu Fri Oct 24 14:55:24 2008 From: bmacgreg from unc.edu (Barbara MacGregor) Date: Fri Oct 24 15:43:11 2008 Subject: phosphatase removal in RNA samples In-Reply-To: <200810241707.m9OH7FV17921@net.bio.net> References: <200810241707.m9OH7FV17921@net.bio.net> Message-ID: <1CE78980-C673-4D1C-8421-AC43CF759472@unc.edu> Hi Simone, Antarctic phosphatase (New England Biolabs) is heat-inactivatible, that might work. Barbara MacGregor > > > From: "Simone Marker" > Date: October 23, 2008 10:53:36 AM EDT > To: methods@net.bio.net > Subject: phosphatase removal in RNA samples > Reply-To: Simone Marker > > > Hi, > > I am looking for chemical properties of short RNA species (siRNA) > and I want > to know wheather they are 5' phosphorylated (e.g. mono-or > triphosphate). I > want to compare their migration behaviour in a denaturing > polyacrylamide gel > (7M urea), which is also influenced by the number of phosphates. I > also need > some RNA samples that are completely dephosphorylated, to compare the > migration. > So I would use calf intestinal alkaline phosphatase (better than SAP > with > RNA), but I do not want to remove the phosphatase by phenol- > chlorophorm > extraction (to much loss of RNA, not quatitative). Do you think that > removal > is necessary? I just want to load the treated samples on the gel > (near the > untreated ones) and see their migration behaviour (in northern). > Does active > phosphatase influence the migration of the RNA in denaturing gels? > (since > CIP is not heat-inactivatable). > > Thank you for your help, > Simone > > From engelbert_buxbaum from hotmail.com Sun Oct 26 12:31:37 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Sun Oct 26 13:22:58 2008 Subject: ppm in mass spectrometry References: Message-ID: Am 23.10.2008, 12:39 Uhr, schrieb Esmaeil Sadroddiny : > Can somebody explain simple definition from ppm in mass spectrometry Are you sure you are talking about MS? ppm usually occurs in the context of nuclear magnetic resonance (NMR) spectroscopy where it denotes the chemical shift of a signal with respect to some reference compound. I.e., if the reference signal is at 400 MHz, a 1 ppm signal would be at 400.0004 MHz. From schisler from gmail.com Mon Oct 27 14:53:15 2008 From: schisler from gmail.com (schisler@gmail.com) Date: Mon Oct 27 16:29:50 2008 Subject: Dynabeads for epitope-tagged protein immuno-precipitation Message-ID: So I am giving the Protein G dynabeads a try for IPs of myc-tagged proteins from mammalian cell lysates. The information that comes with the system if not detailed and there is little technical information to be found on invitrogen's web site. Does anyone routinely use this system for protein immunoprecipitation? I've only tried the 'direct' method so far, but I have not been successful. I've only tried the basic protocol provided, so if someone has a protocol to share, that would be great! Thanks, Jonathan From schisler from gmail.com Mon Oct 27 14:55:34 2008 From: schisler from gmail.com (schisler@gmail.com) Date: Mon Oct 27 16:29:55 2008 Subject: RT-PCR question References: <128edaa6-4367-41e9-ba53-9daa484100e5@e17g2000hsg.googlegroups.com> Message-ID: <24883ca8-d37b-4602-9410-5276641c1657@t41g2000hsc.googlegroups.com> On Oct 24, 6:04?am, chovek69 wrote: > Dear experts, > > I am relatively new to the RT-PCR world and just would like to hear > some suggestions about some basic practices: > > Usually I come up with the RNAs in the evening so what is better: to > run RT-PCR with hexamers overnight (cDNA stay at room temp whole > night) or to freeze the RNA at -80C and do the RT-PCR on the next > day ? > > I am to run PCRs for viral detection (low-copy number) in ticks. > > Thanks If you run your cDNA synthesis reaction using a thermocycler, just add a final hold at 4 degrees and it will be ready for you in the morning. From schisler from gmail.com Mon Oct 27 15:03:19 2008 From: schisler from gmail.com (schisler@gmail.com) Date: Mon Oct 27 16:30:01 2008 Subject: Will SNAP i.d. increade my happiness? References: <6lo27cFd8io4U1@mid.individual.net> Message-ID: <9162fdf9-5875-45e5-87a8-1bb3ab266e64@j22g2000hsf.googlegroups.com> On Oct 16, 1:36?am, Kyle Legate wrote: > TR wrote: > > Dear western-blot experts, > > > A sales rep. from Millipore just left the lab and almost convinced me > > to buy what he calls "a revolution in your western-blot experience": a > > simple device (SNAP i.d.) that makes all incubations shorter by > > forcing all solutions "through" the membrane (using vacuum). > >http://www.millipore.com/immunodetection/id3/snap_id > > > It certainly looks like a much simpler, quicker and cleaner way to do > > all incubation for the westerns, but I would like to hear from people > > who have actually used the mechine. The question is: will the 850 > > euros investment increase our happiness? > > > All the best > > CG > > You will be happy until you have to order new cassettes. The demo unit we tried out for one afternoon (not much time, but that's what they gave us) it worked as well for most antibodies and even better for some. Since we've gotten our new unit, I've not had any success and I am sticking to my old method until I have more time to troubleshoot it. From ijpinto from ibmc.up.pt Tue Oct 28 11:04:51 2008 From: ijpinto from ibmc.up.pt (ijpinto@ibmc.up.pt) Date: Tue Oct 28 11:09:11 2008 Subject: Help with overexpression in Leishmania Message-ID: Dear all, Can anyone help a colleague of mine who's having a serious problem with over-expression of membrane proteins in Leishmania? Thanks a lot! Jorge Dear all, I work with membrane proteins of Leishmania and I have been having some problems. First, does anyone have experience with chimeras? I did a GFP and a c-cmyc fusion protein and none of them could be detected by western blot. Both fusions are C-terminal, though I also did two N-terminal c-myc tagged proteins and again nothing was detected. Second, I tried to produce antibodies against those membrane proteins using recombinant polypeptide and although it recognizes the recombinant truncated protein, it does not recognize in parasite total extracts (in SDS-PAGE). Is this common? Due to this result I introduced an episomic copy of the gene in order to over-express the proteins in the parasites. Since I could not detect the proteins by western blot, I went back to qPCR trying to understand if there were at least higher levels of mRNA. Indeed I saw a fold increase of around 70 for one of the proteins and 4 for another one. The other 2 proteins did not show any increase in the mRNA levels. So, in conclusion, I wonder if the parasites could be able to eliminate the mRNA so fast that no protein is detected and how come the parasites don?t have any mRNA if they carry an episomic copy of the gene inserted in an expression vector. I know this is a lot, but can you help me? I thank you all in advance, T?nia Cruz Iron Genes and Immune System Institute for Molecular and Cell Biology Rua do Campo Alegre, 823 4150-180 Porto Portugal Phone: +351-226074956 email: tcruz@ibmc.up.pt From editor from gene-quantification.info Tue Oct 28 11:14:06 2008 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Tue Oct 28 12:59:38 2008 Subject: qPCR Newsletter - October 2008 Message-ID: <5f8a3dd3-bbba-41e8-8546-c12322bdf79c@x16g2000prn.googlegroups.com> qPCR Newsletter - October 2008 Dear researcher, dear Gene Quantification page reader, Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene Quantification homepage. The focus of this newsletter issue is: - single-cell handling - new page ! - single-cell PCR - pages updated ! - worldwide qPCR application workshops - register now ! - 2nd announcement =3D> qPCR 2009 Event =3D> http://www.qPCR2009.net - online translation service of the Gene Quantification page to: - CHINESE DUTCH FRENCH GERMAN GREEK ITALIAN - JAPANESE KOREAN PORTUGUESE RUSSIAN SPANISH 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/ ---------------------------------------------------------------------------= ----- single-cell handling The purpose of this single-cell handling page is to provide researchers and clinicians with workflows and resources for single-cell molecular analysis such as RT-PCR, quantitative PCR, and sequencing and with a focus on the front-to-backhandling of single cells =3D> http://single-cell-handling.gene-quantification.info Interest in single cell molecular analysis has risen dramatically over the last couple of years, chiefly because single cell molecular analysis is the only way to research genetic heterogeneity, i.e., differences in copy number or gene expression levels between individual cells, or genetically analyze very rare cells such as circulating tumor or fetal cells. Single cell molecular analysis has provided new insight in diverse research areas such as immunology, oncology, stem cells or forensics. However, an often overlooked aspect of single cell analysis is the difficulties of handling single cells for molecular analysis associate with all three major methods for single cell manipulation: - Micromanipulation - LCM =3D Laser Capture Microdissection - Flow cytometry - Publications - Interviews, Talks and Posters ---------------------------------------------------------------------------= ----- Single-cell handling papers - New techniques for isolation of single prokaryotic cells. (REVIEW) - Single cell sorting and cloning. - Sampling efficiency of a single-cell capillary electrophoresis system. - Dynamic single cell culture array. - Quantitative RT-PCR from one single cell using the AmpliGrid technology. - Low volume amplification and sequencing of mitochondrial DNA on a chemically structured chip. - Low-volume amplification on chemically structured chips using the PowerPlex16 DNA amplification kit. - http://single-cell-handling.gene-quantification.info ---------------------------------------------------------------------------= ----- UPDATE single-cell qPCR page The purpose of this single-cell PCR page is to provide researchers with resources (papers, talks, posters) for single-cell molecular analysis such as RT-PCR and quantitative PCR =3D> http://singlecell.gene-quantification.info/ New and innovative papers around single-cell qPCR: Intracellular expression profiles measured by real-time PCR tomography in the Xenopus laevis oocyte. Quantification of mRNA in single cells and modelling of RT-qPCR induced noise. Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. 220-plex microRNA expression profile of a single cell. MicroRNA quantitation from a single cell by PCR using SYBR Green detection and LNA-based primers. Split single-cell RT-PCR analysis of Purkinje cells. The real-time polymerase chain reaction (a section with single-cell qRT-PCR). Single-cell gene expression profiling. Gene expression and the myth of the average cell. Combining laser capture microdissection with quantitative real-time PCR: effects of tissue manipulation on RNA quality and gene expression. RNA amplification strategies for small sample populations. Gene expression of single chondrocytes. Gene expression profiling of individual bovine nuclear transfer blastocysts. Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood. Expression profiling of small cellular samples in cancer: less is more. ---------------------------------------------------------------------------= ----- 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://qPCR2009.net download event FLYER =3D> http://www.bioeps.com/qpcr2009/qPCR-2009-2nd-an= nouncement.pdf It is a pleasure to announce the Nobel Prize Laureate Kary Mullis at the qPCR 2009 event in an own session =9325th Anniversary of PCR=94 List of confirmed speakers =3D> http://speakers.qpcr2009.net/ An industrial exhibition with the 30 world leading companies will be held during the qPCR Symposium March 9-11 =3D> http://exhibition.qpcr2009= .net/ Our sponsors =3D> http://sponsors.qpcr2009.net/ Please register =3D> http://registration.qpcr2009.net/ ---------------------------------------------------------------------------= ----- qPCR WORKSHOP BioEPS GmbH / TATAA Biocenter Germany - qPCR Application workshops At the TATAA Biocenter Germany we offer qPCR application workshops, a 3-day qPCR Core Module and a 2-day qPCR Biostatistics Module. All courses are held regularly in G=F6teborg, Sweden, in English and in Freising-Weihenstephan, Germany, in German and English, and in Prague, Czech Republic in English and Czech. Depending on the occasion the workshop language and the different prices may apply. Further customized workshops and specialized trainings will be held as well across Europe and world-wide. TATAA Biocenter Germany workshops are held in cooperation with BioEPS GmbH, located at the campus of the Technical University of Munich, in Freising-Weihenstephan, very close to the Munich Airport (MUC). For more information and registration, please see our web page =3D> http://TATAA.gene-quantification.info/ Course Occasions 2008/2009: 3-day qPCR Core Module (Mon. - Wed.) 2-day BioStatistics Module (Thu. - Fri.) single-cell qPCR Module (Mon. - Wed.) 27-31 Oct G=F6teborg qPCR Core Module + HRM + Biostatistics 17-21 Nov Prague qPCR Core Module + Practical Biostatistics 24-28 Nov Freising Germany qPCR Core Module + Biostatistics (English language) 1-5 Dec G=F6teborg qPCR Core Module + Biostatistics 8 - 10 December 2008 (E) NEW singel-cell qPCR Application Workshop 15-19 Dec Prague RNA Isolation + Expression Profiling and Data Analysis 26 - 30th January 2009 in Freising, Germany, English language ---------------------------------------------------------------------------= ----- Forward Please send the qPCR NEWS to further scientists and friends who are interested in qPCR ! Best regards, Michael W. 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Disclaimer & Copyrights are displayed on the homepage www.gene-quantification.com From daniela.schwoerer from googlemail.com Wed Oct 29 04:51:57 2008 From: daniela.schwoerer from googlemail.com (Daniela Schwoerer) Date: Wed Oct 29 11:15:52 2008 Subject: Problems using pGEX - other expression vector? Message-ID: <5696f7c50810290251m52aa93c8we751d636b59bec67@mail.gmail.com> Hi everyone, I tried to express a protein using pGEX-4T-3 vector which provides a tag for GST-fusion proteins. Unfortunately it doesn't seem to work and also technical support is not helpful. I tried everything, I believe the multicloningsite ist mutated. Or its the moon's fault, who knows. Does anyone use this vector and did you also have problems when trying to clone a gene? (i used BamHI and XhoI restriction sites) Does anyone have an idea which other vector i could use, most likely also with a GST-tag for fusion proteins? Thanks for the help! From sudhee26 from gmail.com Thu Oct 30 03:18:20 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu Oct 30 11:42:48 2008 Subject: Genes from Minus strand Message-ID: Hi,Most of the genes are transcribed from positive strand. But there are many genes which are transcribed-translated-from minus strand. Can anybody give me resources and where i can find more information about such genes! how to find all human genes which are transcribed from minus strand? also i have little confusion about "antisense strand transcripts" and "genes from minus strand".. any links are welcome. i really need to understand this stuff..so help me. -- Think before agree Think before you nod STOP thinking to be a God From Nikola.Wenta from nottingham.ac.uk Thu Oct 30 18:01:50 2008 From: Nikola.Wenta from nottingham.ac.uk (Nikola Wenta) Date: Fri Oct 31 13:12:03 2008 Subject: Problems using pGEX - other expression vector? In-Reply-To: <200810301704.m9UH49V01467@net.bio.net> References: <200810301704.m9UH49V01467@net.bio.net> Message-ID: Hi! Maybe it's codon usage? Then you should use a codon-optimized strain like Rosetta. If it's the vector, why not trying pASK-IBA; they also provide the same restriction sites, and they provide a Strep-tag which makes protein easier and yields in more pure than GST. Cheers Niko 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.