From virashkgupta from gmail.com Sat Aug 1 01:46:05 2009 From: virashkgupta from gmail.com (Virash Gupta) Date: Sat Aug 1 11:18:20 2009 Subject: Methods Digest, Vol 50, Issue 22- need to identify a contaminant in the cell culture Message-ID: What is your interest: contaminant or the cell culture. Define the cell culture is it a bacterial culture or tissue culture. I would suggest to concentrate on culture than your contaminant. On 7/30/09, 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: storing of enzyme (Dr. Hiranya S. Roychowdhury) > 2. Re: guanidinium disposal (camjay) > 3. need to identify a contaminant in the cell culture > (Sudheendra Rao N R) > 4. About SSCP (Marcos Moraes) > 5. reconstitution of membrane proteins (Carmen P Pena Diaz) > 6. Re: About SSCP (Glenn Dunshea) > 7. phmeter (Paola Contreras) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 29 Jul 2009 09:26:15 -0600 (MDT) > From: "Dr. Hiranya S. Roychowdhury" > Subject: Re: storing of enzyme > To: "Nick Theodorakis" > Cc: methods@magpie.bio.indiana.edu > Message-ID: <1603.128.123.174.0.1248881175.squirrel@webmail.nmsu.edu> > Content-Type: text/plain;charset=3Diso-8859-1 > > Mostly cellulose nitrate. I had done it "way back when ..." using these. > > > > On Jul 28, 3:51 pm, "Dr. Hiranya S. Roychowdhury" > > wrote: > >> Use a buffer (10mM Tris-EDTA, pH 6.5 to 7.2, or 50mM phosphate buffer > >> should work) and add glycerol to 35% (as high as 50%). Store in a -20= C > >> freezer (no auto defrost). > >> > >> Cellulase can also be stores freeze dried after the appropriate dialys= is > >> against distilled water containing 1mM DTT. > >> > > > > Is dialysis tubing still made from cellulose? > > > > Nick > > > > -- > > Nick Theodorakis > > nick_theodorakis@hotmail.com > > contact form: > > http://theodorakis.net/contact.html > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > -- > Hiranya S. Roychowdhury, Ph.D. > Asst. Professor, > Health & Public Services > Dona Ana Community College > New Mexico State University > Las Cruces, NM 88003 > > > > ------------------------------ > > Message: 2 > Date: Wed, 29 Jul 2009 08:26:38 -0700 (PDT) > From: camjay > Subject: Re: guanidinium disposal > To: methods@net.bio.net > Message-ID: > > Content-Type: text/plain; charset=3DISO-8859-1 > > On 28 July, 23:25, "Laurel Cooper" wrote: > > I am cleaning out a bunch of old stuff in the lab I am in and there are > > numerous bottles of guanidinium isothiocyanate and guanidinium chloride > > solutions. Can these be disposed of down the drain with lots of water = or > do > > they need to go to waste disposal. They are solutions from commercial > kits. > > Here in Europe guanidinium isothiocyanate is rated "Harmful to aquatic > organisms" and "May cause long-term adverse effects in the aquatic > environment" and I certainly wouldn't dispose of any down the drain. > > Really you should seek local guidance from your institute, water > supply or sewerage company or appropriate regulatory authority. > > > ------------------------------ > > Message: 3 > Date: Thu, 30 Jul 2009 00:37:45 +0530 > From: Sudheendra Rao N R > Subject: need to identify a contaminant in the cell culture > To: methods@magpie.bio.indiana.edu, methods > > Message-ID: > > Content-Type: text/plain; charset=3DISO-8859-1 > > Hi, all,I recently found this contaminant in one of the thawed vials..i > want > to ID it.i dont know how to post pictures in this list..but i will give > description.. > > 1. macroscopically: it appears floating mass of myecilium: two > latyers..layer 1-close to cell surface sometimes sticking to cells..layer > two: surface of media..looks like oil has spilled in media..looks > greasy..very difficult to get rid of even after repeated PBS > washings..layer > 1: is clump of mycelia..layer 2: looks like broken mycelia floating..both > layer1 and layer 2 are not motile. > > 2. cell morphology does not seem to change a lot..cells look healthy.but > they die suddenly when the contaminant number flourishes. no change in > media > color except media top looks greasy/oily..imagine oil spilled in > sea!!!..they grow in FBS+pen-strep, they are resistant to geneticin too.. > > 3. i took the supernatant and plated on LB agar (thats the only thing i > have--i dont do microbiology :))..with ampicillin (since i found out that > this is penicillin resistant)..within 6 days i small colonies which > increased up to 3 mm diameter by 15 days..all colonies have same > morphology..i guess hence its not something spurious..and also on two > different plates i got same kind of colonies. > > 4. > Form: Circular (grossly, but when looked at 10X magnification we can make > out that its not so) > Color: Cream > Margin: undulating > Elevation: umbonate > Surface: smooth. Macroscopically surface looks infrequently studdent with > cream colored spekles. > Opacity: Opaque > > 5. 40X magnification revealed very interestingly margin of this colony > which > appeared on LB plate with ampicillin had Claw like projection > around..covering almost entire periphery (around) of the colony..probably > thats how colony spreads and becomes big..also at 40X the colony does not > look homogenous..its made of fine grains.. > > so what is it..I will try posting some pics to my blog.. > > meanwhile..i really appreciate the ID..so that I can get rid of it..i am > anyway ordering amphotericin B. > > regards > sudheendra. > > > > -- > Think before agree > Think before you nod > STOP thinking > to be a God > > > ------------------------------ > > Message: 4 > Date: Wed, 29 Jul 2009 16:49:14 -0700 (PDT) > From: Marcos Moraes > Subject: About SSCP > To: methods@magpie.bio.indiana.edu > Message-ID: <204388.64103.qm@web110313.mail.gq1.yahoo.com> > Content-Type: text/plain; charset=3Diso-8859-1 > > Dear Bruno, > > Surfing on net I found a message ( > http://www.bio.net/bionet/mm/methods/2002-February/092258.html) that you > said things about SSCP resolution. In that moment, you said you was runni= ng > 1XTBE + 10% glycerol gels. How many conditions have you ever tested? I'm > asking because after a long time screening for mutation for COL1A1 gene > (Osteogenesis imperfecta), i realized that the low ratio of mutants could= be > the SSCP conditions. Here, the SSCP runs at room temperature (PAGE-SSCP S= 2 > Biometra) and at the moment, I'm trying to figure out a condition(s) capa= ble > to detect the mutations. I'm testing absence, 5% and 10% glycerol; no > glycerol + 10% sucrose; MDE 0.5X w/ or w/out glycerol 5%, all of them wit= h > 0.5X TBE. Do you would recommend me to use 1X TBE instead of 0.5X? Does i= t > make any difference for detection? > > Best regards, > > Marcos Moraes| Mestrando em Biotecnologia | Sa=FAde > Laborat=F3rio de Gen=E9tica Humana e Molecular > > N=FAcleo de Gen=E9tica Humana e Molecular | Depto. Ci=EAncias Biol=F3gica= s | Centro > de Ci=EAncias Humanas e Naturais | Universidade Federal do Esp=EDrito San= to > Av. Marechal Campos, 1468 - Maru=EDpe - 29040-090 - Vit=F3ria - ES - Bras= il > T +55 27 3335 7253 | C+55 27 9249 0190 | markydor@yahoo.com.br > http://lattes.cnpq.br/0799432282422121 > > > > _________________________________________________________________________= ___________ > Veja quais s=E3o os assuntos do momento no Yahoo! +Buscados > http://br.maisbuscados.yahoo.com > > ------------------------------ > > Message: 5 > Date: Thu, 30 Jul 2009 16:47:14 +0100 > From: "Carmen P Pena Diaz" > Subject: reconstitution of membrane proteins > To: > Message-ID: > <445800D8FD5D3D43878FD7D2842A304743542C@EXCL2VS2.adir.hull.ac.uk> > Content-Type: text/plain; charset=3D"iso-8859-1" > > > Hello > > > I was wondering how many of you work with membrane proteins and might me > able to help out with this one. > > > I'm trying to reconstitute mitochondrial carriers into liposomes. The > established protocol is done in 3 major steps: > > a) isolating the protein, of course, from bacterial strain after > expression. the isolation is basically from inclusion bodies and > solubilization in 1% sarkosyl in tris buffer. > > b) making the proteoliposomes, which is done incorporating the protein in > extruded egg yolk phospholipids (9mg/mL), internal substrate (ADP or ATP = in > my case, 30mM), Tx-114 and cardiolipin in 10mM buffer MOPS, 50mM NaCl. t= his > mix is passed through a Amberlite XAD-2 column, 24X > > the un-incorporated internal substrate is eliminated using another column= , > Dowex AG1-X8, equilibrated with the same buffer MOPS as before > > c) the transport assay is done mixing the liposomes with ATP (3H) and > stoping the reaction with 30mM pyridoxal 5=92 phosphate and 10mM > bathophenanthroline after 0,2,5 & 10minutes. > > excess of non-trasported radiactivity is eliminated using sephadex G-75 > equilibrated with the same Mops buffer as before. > > > So far, no transport has been observed. I was wondering if any of you cou= ld > shed some light on the subject. > > > regards > > > Priscila > -------------- next part -------------- > > *************************************************************************= **************** > To view the terms under which this email is distributed, please go to > http://www.hull.ac.uk/legal/email_disclaimer.html > > *************************************************************************= **************** > > ------------------------------ > > Message: 6 > Date: Thu, 30 Jul 2009 13:34:45 +0800 > From: Glenn Dunshea > Subject: Re: About SSCP > To: Marcos Moraes > Cc: methods@magpie.bio.indiana.edu > Message-ID: > <8acca5110907292234n192e2404t1585c53ce58d2b28@mail.gmail.com> > Content-Type: text/plain; charset=3DISO-8859-1 > > I have found MDE solution excellent for detection of mutations without th= e > need for glycerol. Something to consider when running your trials is that > one of the most critical parameters for SSCP assays is constant > temperature. > I would not recommend running SSCP at room temperature.. > I hope your experiments are successful. > Cheers, > Glenn > > On Thu, Jul 30, 2009 at 7:49 AM, Marcos Moraes >wrote: > > > Dear Bruno, > > > > Surfing on net I found a message ( > > http://www.bio.net/bionet/mm/methods/2002-February/092258.html) that > > you said things about SSCP resolution. In that moment, you said you was > > running 1XTBE + 10% glycerol gels. How many conditions have you ever > tested? > > I'm asking because after a long time screening for mutation for COL1A1 > gene > > (Osteogenesis imperfecta), i realized that the low ratio of mutants cou= ld > be > > the SSCP conditions. Here, the SSCP runs at room temperature (PAGE-SSCP > S2 > > Biometra) and at the moment, I'm trying to figure out a condition(s) > capable > > to detect the mutations. I'm testing absence, 5% and 10% glycerol; no > > glycerol + 10% sucrose; MDE 0.5X w/ or w/out glycerol 5%, all of them > with > > 0.5X TBE. Do you would recommend me to use 1X TBE instead of 0.5X? Does > it > > make any difference for detection? > > > > Best regards, > > > > Marcos Moraes| Mestrando em Biotecnologia | Sa=FAde > > Laborat=F3rio de Gen=E9tica Humana e Molecular > > > > N=FAcleo de Gen=E9tica Humana e Molecular | Depto. Ci=EAncias Biol=F3gi= cas | > Centro > > de Ci=EAncias Humanas e Naturais | Universidade Federal do Esp=EDrito S= anto > > Av. Marechal Campos, 1468 - Maru=EDpe - 29040-090 - Vit=F3ria - ES - Br= asil > > T +55 27 3335 7253 | C+55 27 9249 0190 | markydor@yahoo.com.br > > http://lattes.cnpq.br/0799432282422121 > > > > > > > > > ______________________________________________________________________= ______________ > > Veja quais s=E3o os assuntos do momento no Yahoo! +Buscados > > http://br.maisbuscados.yahoo.com > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > ------------------------------ > > Message: 7 > Date: Thu, 30 Jul 2009 11:48:29 -0300 > From: Paola Contreras > Subject: phmeter > To: methods@magpie.bio.indiana.edu > Message-ID: > <9df709230907300748ra724e4j77176a1fb3b87b7e@mail.gmail.com> > Content-Type: text/plain; charset=3DISO-8859-1 > > I found in the methods mailing list the following: We bought a Sentron > model 1001. that is why I thought you can help me since in our lab we > have the same phmeter but without the corresponding instructions for > users. I would appreciate if someone could give me an idea on how to > get the users' manual. I have already asked the makers but did not get > an answer. > best regards, > Paola. > > -- > Paola Contreras, MSc > Departamento de Fisiolog=EDa > Facultad de Medicina > Universidad de la Rep=FAblica > Montevideo, URUGUAY > Tel: 598 2 924 94 53 > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 50, Issue 22 > *************************************** > --=20 Dr V K Gupta Sr Microbiologist (Molecular Biology) Insect Molecular Biology Lab Department of Entomology Punjab Agricultural University Ludhiana (Pb)-141004- India M: 09815963210 From sudhee26 from gmail.com Sun Aug 2 08:24:39 2009 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Sun Aug 2 12:02:46 2009 Subject: Methods Digest, Vol 50, Issue 22- need to identify a contaminant in the cell culture In-Reply-To: References: Message-ID: Dear Viresh,Thanks for a response. We have already taken measures that woul= d ensure healthy cells. I want to stress that my mail does mention "cells" "FBS" and things like that which would be pointing towards "cell culture" i was talking of. regarding contamination in that..and identifying it..i posted specifically to get ID of it..since you are microbiologist yourself you would probably interested in stuffs like these..it was out of curiosity i posted it on methods and not out of sheer frustration (i did get frustrated because it was quite a difficult thing to get rid off..but we just threw everything an= d things seem to be fine now). I appreciate your concern again. sincerely sudheendra. On Sat, Aug 1, 2009 at 12:16 PM, Virash Gupta wrote= : > What is your interest: contaminant or the cell culture. Define the cell > culture is it a bacterial culture or tissue culture. I would suggest to > concentrate on culture than your contaminant. > > On 7/30/09, 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: storing of enzyme (Dr. Hiranya S. Roychowdhury) > > 2. Re: guanidinium disposal (camjay) > > 3. need to identify a contaminant in the cell culture > > (Sudheendra Rao N R) > > 4. About SSCP (Marcos Moraes) > > 5. reconstitution of membrane proteins (Carmen P Pena Diaz) > > 6. Re: About SSCP (Glenn Dunshea) > > 7. phmeter (Paola Contreras) > > > > > > ---------------------------------------------------------------------- > > > > Message: 1 > > Date: Wed, 29 Jul 2009 09:26:15 -0600 (MDT) > > From: "Dr. Hiranya S. Roychowdhury" > > Subject: Re: storing of enzyme > > To: "Nick Theodorakis" > > Cc: methods@magpie.bio.indiana.edu > > Message-ID: <1603.128.123.174.0.1248881175.squirrel@webmail.nmsu.edu> > > Content-Type: text/plain;charset=3Diso-8859-1 > > > > Mostly cellulose nitrate. I had done it "way back when ..." using thes= e. > > > > > > > On Jul 28, 3:51 pm, "Dr. Hiranya S. Roychowdhury" > > > wrote: > > >> Use a buffer (10mM Tris-EDTA, pH 6.5 to 7.2, or 50mM phosphate buffe= r > > >> should work) and add glycerol to 35% (as high as 50%). Store in a > -20C > > >> freezer (no auto defrost). > > >> > > >> Cellulase can also be stores freeze dried after the appropriate > dialysis > > >> against distilled water containing 1mM DTT. > > >> > > > > > > Is dialysis tubing still made from cellulose? > > > > > > Nick > > > > > > -- > > > Nick Theodorakis > > > nick_theodorakis@hotmail.com > > > contact form: > > > http://theodorakis.net/contact.html > > > _______________________________________________ > > > Methods mailing list > > > Methods@net.bio.net > > > http://www.bio.net/biomail/listinfo/methods > > > > > > > > > -- > > Hiranya S. Roychowdhury, Ph.D. > > Asst. Professor, > > Health & Public Services > > Dona Ana Community College > > New Mexico State University > > Las Cruces, NM 88003 > > > > > > > > ------------------------------ > > > > Message: 2 > > Date: Wed, 29 Jul 2009 08:26:38 -0700 (PDT) > > From: camjay > > Subject: Re: guanidinium disposal > > To: methods@net.bio.net > > Message-ID: > > < > cd0680b4-7644-461b-91a6-c4815e4b37c3@g31g2000yqc.googlegroups.com> > > Content-Type: text/plain; charset=3DISO-8859-1 > > > > On 28 July, 23:25, "Laurel Cooper" wrote: > > > I am cleaning out a bunch of old stuff in the lab I am in and there a= re > > > numerous bottles of guanidinium isothiocyanate and guanidinium chlori= de > > > solutions. Can these be disposed of down the drain with lots of wate= r > or > > do > > > they need to go to waste disposal. They are solutions from commercia= l > > kits. > > > > Here in Europe guanidinium isothiocyanate is rated "Harmful to aquatic > > organisms" and "May cause long-term adverse effects in the aquatic > > environment" and I certainly wouldn't dispose of any down the drain. > > > > Really you should seek local guidance from your institute, water > > supply or sewerage company or appropriate regulatory authority. > > > > > > ------------------------------ > > > > Message: 3 > > Date: Thu, 30 Jul 2009 00:37:45 +0530 > > From: Sudheendra Rao N R > > Subject: need to identify a contaminant in the cell culture > > To: methods@magpie.bio.indiana.edu, methods > > > > Message-ID: > > > > Content-Type: text/plain; charset=3DISO-8859-1 > > > > Hi, all,I recently found this contaminant in one of the thawed vials..i > > want > > to ID it.i dont know how to post pictures in this list..but i will give > > description.. > > > > 1. macroscopically: it appears floating mass of myecilium: two > > latyers..layer 1-close to cell surface sometimes sticking to cells..lay= er > > two: surface of media..looks like oil has spilled in media..looks > > greasy..very difficult to get rid of even after repeated PBS > > washings..layer > > 1: is clump of mycelia..layer 2: looks like broken mycelia floating..bo= th > > layer1 and layer 2 are not motile. > > > > 2. cell morphology does not seem to change a lot..cells look healthy.bu= t > > they die suddenly when the contaminant number flourishes. no change in > > media > > color except media top looks greasy/oily..imagine oil spilled in > > sea!!!..they grow in FBS+pen-strep, they are resistant to geneticin too= .. > > > > 3. i took the supernatant and plated on LB agar (thats the only thing i > > have--i dont do microbiology :))..with ampicillin (since i found out th= at > > this is penicillin resistant)..within 6 days i small colonies which > > increased up to 3 mm diameter by 15 days..all colonies have same > > morphology..i guess hence its not something spurious..and also on two > > different plates i got same kind of colonies. > > > > 4. > > Form: Circular (grossly, but when looked at 10X magnification we can ma= ke > > out that its not so) > > Color: Cream > > Margin: undulating > > Elevation: umbonate > > Surface: smooth. Macroscopically surface looks infrequently studdent wi= th > > cream colored spekles. > > Opacity: Opaque > > > > 5. 40X magnification revealed very interestingly margin of this colony > > which > > appeared on LB plate with ampicillin had Claw like projection > > around..covering almost entire periphery (around) of the colony..probab= ly > > thats how colony spreads and becomes big..also at 40X the colony does n= ot > > look homogenous..its made of fine grains.. > > > > so what is it..I will try posting some pics to my blog.. > > > > meanwhile..i really appreciate the ID..so that I can get rid of it..i a= m > > anyway ordering amphotericin B. > > > > regards > > sudheendra. > > > > > > > > -- > > Think before agree > > Think before you nod > > STOP thinking > > to be a God > > > > > > ------------------------------ > > > > Message: 4 > > Date: Wed, 29 Jul 2009 16:49:14 -0700 (PDT) > > From: Marcos Moraes > > Subject: About SSCP > > To: methods@magpie.bio.indiana.edu > > Message-ID: <204388.64103.qm@web110313.mail.gq1.yahoo.com> > > Content-Type: text/plain; charset=3Diso-8859-1 > > > > Dear Bruno, > > > > Surfing on net I found a message ( > > http://www.bio.net/bionet/mm/methods/2002-February/092258.html) that yo= u > > said things about SSCP resolution. In that moment, you said you was > running > > 1XTBE + 10% glycerol gels. How many conditions have you ever tested? I'= m > > asking because after a long time screening for mutation for COL1A1 gene > > (Osteogenesis imperfecta), i realized that the low ratio of mutants cou= ld > be > > the SSCP conditions. Here, the SSCP runs at room temperature (PAGE-SSCP > S2 > > Biometra) and at the moment, I'm trying to figure out a condition(s) > capable > > to detect the mutations. I'm testing absence, 5% and 10% glycerol; no > > glycerol + 10% sucrose; MDE 0.5X w/ or w/out glycerol 5%, all of them > with > > 0.5X TBE. Do you would recommend me to use 1X TBE instead of 0.5X? Does > it > > make any difference for detection? > > > > Best regards, > > > > Marcos Moraes| Mestrando em Biotecnologia | Sa=FAde > > Laborat=F3rio de Gen=E9tica Humana e Molecular > > > > N=FAcleo de Gen=E9tica Humana e Molecular | Depto. Ci=EAncias Biol=F3gi= cas | > Centro > > de Ci=EAncias Humanas e Naturais | Universidade Federal do Esp=EDrito S= anto > > Av. Marechal Campos, 1468 - Maru=EDpe - 29040-090 - Vit=F3ria - ES - Br= asil > > T +55 27 3335 7253 | C+55 27 9249 0190 | markydor@yahoo.com.br > > http://lattes.cnpq.br/0799432282422121 > > > > > > > > > _________________________________________________________________________= ___________ > > Veja quais s=E3o os assuntos do momento no Yahoo! +Buscados > > http://br.maisbuscados.yahoo.com > > > > ------------------------------ > > > > Message: 5 > > Date: Thu, 30 Jul 2009 16:47:14 +0100 > > From: "Carmen P Pena Diaz" > > Subject: reconstitution of membrane proteins > > To: > > Message-ID: > > <445800D8FD5D3D43878FD7D2842A304743542C@EXCL2VS2.adir.hull.ac.uk= > > > Content-Type: text/plain; charset=3D"iso-8859-1" > > > > > > Hello > > > > > > I was wondering how many of you work with membrane proteins and might m= e > > able to help out with this one. > > > > > > I'm trying to reconstitute mitochondrial carriers into liposomes. The > > established protocol is done in 3 major steps: > > > > a) isolating the protein, of course, from bacterial strain after > > expression. the isolation is basically from inclusion bodies and > > solubilization in 1% sarkosyl in tris buffer. > > > > b) making the proteoliposomes, which is done incorporating the protein = in > > extruded egg yolk phospholipids (9mg/mL), internal substrate (ADP or AT= P > in > > my case, 30mM), Tx-114 and cardiolipin in 10mM buffer MOPS, 50mM NaCl. > this > > mix is passed through a Amberlite XAD-2 column, 24X > > > > the un-incorporated internal substrate is eliminated using another > column, > > Dowex AG1-X8, equilibrated with the same buffer MOPS as before > > > > c) the transport assay is done mixing the liposomes with ATP (3H) and > > stoping the reaction with 30mM pyridoxal 5=92 phosphate and 10mM > > bathophenanthroline after 0,2,5 & 10minutes. > > > > excess of non-trasported radiactivity is eliminated using sephadex G-75 > > equilibrated with the same Mops buffer as before. > > > > > > So far, no transport has been observed. I was wondering if any of you > could > > shed some light on the subject. > > > > > > regards > > > > > > Priscila > > -------------- next part -------------- > > > > > *************************************************************************= **************** > > To view the terms under which this email is distributed, please go to > > http://www.hull.ac.uk/legal/email_disclaimer.html > > > > > *************************************************************************= **************** > > > > ------------------------------ > > > > Message: 6 > > Date: Thu, 30 Jul 2009 13:34:45 +0800 > > From: Glenn Dunshea > > Subject: Re: About SSCP > > To: Marcos Moraes > > Cc: methods@magpie.bio.indiana.edu > > Message-ID: > > <8acca5110907292234n192e2404t1585c53ce58d2b28@mail.gmail.com> > > Content-Type: text/plain; charset=3DISO-8859-1 > > > > I have found MDE solution excellent for detection of mutations without > the > > need for glycerol. Something to consider when running your trials is th= at > > one of the most critical parameters for SSCP assays is constant > > temperature. > > I would not recommend running SSCP at room temperature.. > > I hope your experiments are successful. > > Cheers, > > Glenn > > > > On Thu, Jul 30, 2009 at 7:49 AM, Marcos Moraes > >wrote: > > > > > Dear Bruno, > > > > > > Surfing on net I found a message ( > > > http://www.bio.net/bionet/mm/methods/2002-February/092258.html) that > > > you said things about SSCP resolution. In that moment, you said you w= as > > > running 1XTBE + 10% glycerol gels. How many conditions have you ever > > tested? > > > I'm asking because after a long time screening for mutation for COL1A= 1 > > gene > > > (Osteogenesis imperfecta), i realized that the low ratio of mutants > could > > be > > > the SSCP conditions. Here, the SSCP runs at room temperature (PAGE-SS= CP > > S2 > > > Biometra) and at the moment, I'm trying to figure out a condition(s) > > capable > > > to detect the mutations. I'm testing absence, 5% and 10% glycerol; no > > > glycerol + 10% sucrose; MDE 0.5X w/ or w/out glycerol 5%, all of them > > with > > > 0.5X TBE. Do you would recommend me to use 1X TBE instead of 0.5X? Do= es > > it > > > make any difference for detection? > > > > > > Best regards, > > > > > > Marcos Moraes| Mestrando em Biotecnologia | Sa=FAde > > > Laborat=F3rio de Gen=E9tica Humana e Molecular > > > > > > N=FAcleo de Gen=E9tica Humana e Molecular | Depto. Ci=EAncias Biol=F3= gicas | > > Centro > > > de Ci=EAncias Humanas e Naturais | Universidade Federal do Esp=EDrito= Santo > > > Av. Marechal Campos, 1468 - Maru=EDpe - 29040-090 - Vit=F3ria - ES - = Brasil > > > T +55 27 3335 7253 | C+55 27 9249 0190 | markydor@yahoo.com.br > > > http://lattes.cnpq.br/0799432282422121 > > > > > > > > > > > > > > > ________________________________________________________________________= ____________ > > > Veja quais s=E3o os assuntos do momento no Yahoo! +Buscados > > > http://br.maisbuscados.yahoo.com > > > _______________________________________________ > > > Methods mailing list > > > Methods@net.bio.net > > > http://www.bio.net/biomail/listinfo/methods > > > > > > > > > ------------------------------ > > > > Message: 7 > > Date: Thu, 30 Jul 2009 11:48:29 -0300 > > From: Paola Contreras > > Subject: phmeter > > To: methods@magpie.bio.indiana.edu > > Message-ID: > > <9df709230907300748ra724e4j77176a1fb3b87b7e@mail.gmail.com> > > Content-Type: text/plain; charset=3DISO-8859-1 > > > > I found in the methods mailing list the following: We bought a Sentron > > model 1001. that is why I thought you can help me since in our lab we > > have the same phmeter but without the corresponding instructions for > > users. I would appreciate if someone could give me an idea on how to > > get the users' manual. I have already asked the makers but did not get > > an answer. > > best regards, > > Paola. > > > > -- > > Paola Contreras, MSc > > Departamento de Fisiolog=EDa > > Facultad de Medicina > > Universidad de la Rep=FAblica > > Montevideo, URUGUAY > > Tel: 598 2 924 94 53 > > > > > > > > ------------------------------ > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > End of Methods Digest, Vol 50, Issue 22 > > *************************************** > > > > > > -- > Dr V K Gupta > Sr Microbiologist (Molecular Biology) > Insect Molecular Biology Lab > Department of Entomology > Punjab Agricultural University > Ludhiana (Pb)-141004- India > M: 09815963210 > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > --=20 Think before agree Think before you nod STOP thinking to be a God From paulslpool from yahoo.co.uk Sun Aug 2 22:17:55 2009 From: paulslpool from yahoo.co.uk (Paul smith) Date: Sun Aug 2 22:26:58 2009 Subject: Seeking method for fatty acid content determination in plant seeds and seedlings Message-ID: <64675.89182.qm@web26404.mail.ukl.yahoo.com> Hello, Not being a chemist, I am seeking a detailed protocol for extraction and GC analysis of "free" fatty acids and fatty acids in triglycerides (TGAs) in Arabidopsis seeds and seedlings (time-course experiments). This is to look quantitatively and qualitatively at the amount and composition of TGAs in seeds of a mutant vs. wildtype and their changes during germination and seedling establishment. None of the methods I have seen in the literature is detailed enough for someone inexperienced to follow without assistance of an expert (none available where I work). I am also concerned about the safety aspects with all the dangerous organic chemicals involved. I would be very grateful to any expert that may provide me with a detailed protocol that can safely be followed. I would also appreciate that details are included for special glassware & equipment that is required for the procedures. Please note that I do have access to a Agilent 6890N GC machine with an automatic injector and a 30 m HP-5 30 m column, suitable for FAs. Thank you! Paul Smith paulslpool@yahoo.co.uk From Lionel.Brooks from dartmouth.edu Mon Aug 3 11:41:49 2009 From: Lionel.Brooks from dartmouth.edu (Lionel Brooks 3rd) Date: Mon Aug 3 12:43:57 2009 Subject: Ab smear obscures signal on IP-western Message-ID: <4A77134D.7030404@dartmouth.edu> Hello all, I am performing IP-westerns with affinity-purified antibody as the affinity reagent. The procedure must be performed under non-reducing conditions. Unfortunately, when I exclude DTT from my sample loading buffer then all I can see on the Western is a big Ab smear that runs from ~210 kD to ~20 kD. I suspect that the Ab may be partially reduced because there is 1 mM DTT in my lysis buffer. However I am not sure about this. Does anyone have any ideas about how I might salvage these samples? Is there any way to make my Ab stay at the top of the gel? thanks, Lee From cjmcdermottroe from googlemail.com Tue Aug 4 08:08:45 2009 From: cjmcdermottroe from googlemail.com (Chris McDermott-Roe) Date: Tue Aug 4 11:43:23 2009 Subject: fusion protein purification problem Message-ID: Hi all, I have produced a myc-conjugated fusion protein and hope to express it in mammalian cells and subsequently purify it. Are there any myc resins out there that would permit large-scale purification of my protein? Second, if I was to transfect my protein into say HEK293 cells, would I need to worry about co-purifying endogenous m-Myc if using such a resin? Thanks all Chris From kuforen from gmail.com Mon Aug 3 21:02:27 2009 From: kuforen from gmail.com (Seong Hwan Park) Date: Tue Aug 4 11:43:36 2009 Subject: RNA extraction from blood using Chelex 100 resin? Message-ID: <7e2b93f90908031902m7461ab77x8211f14c58d2a743@mail.gmail.com> Hi, guys. Does anybody have an experience with RNA extraction from blood using Chelex 100 resin? Because I have a DNA version of Chelex 100 resin protocol, I modified it. 1. Whole blood 250ul + cell(RBC) lysis buffer 375ul -> 4'C incubation -> centrifuge -> discard supernatant 2. repeat 1 twice more 3. apply 100ul of 5% Chelex 100 resin to the pellet 4. boiling for 20min 5. centrifuge and collect supernatant 6. DNase I treatment for supernatant and heat inactivation of DNase I 7. RT-PCR Do you think this protocol work? If you have any comment on it, please let me know. Seong -- Seong Hwan Park, M.D., Ph.D. Instructor in Legal Medicine, College of Medicine, Korea University 126-1 Anadong 5ga, Seongbukgu, Seoul 136-704 South Korea E-mail: kuforen@gmail.com or pelvis@korea.ac.kr Tel: 82-2-920-6158 Fax: 82-2-928-3901 From sanja.ovens from police.vic.gov.au Mon Aug 3 23:53:53 2009 From: sanja.ovens from police.vic.gov.au (Ovens, Sanja) Date: Tue Aug 4 11:43:41 2009 Subject: An effective method for elimination of DNA and RNA residues Message-ID: Hi Could I have a bit more information on this method? Thank you Kind regards Sanja Ovens Victoria Police Forensic Services Department Biology - DNA 31 Forensic Dve Macleod Victoria 3085 Australia Email: sanja.ovens@police.vic.gov.au ================================================================================================ EMAIL DISCLAIMER This email and any attachments are confidential. They may also be subject to copyright. If you are not an intended recipient of this email please immediately contact us by replying to this email and then delete this email. You must not read, use, copy, retain, forward or disclose this email or any attachment. We do not accept any liability arising from or in connection with unauthorised use or disclosure of the information contained in this email or any attachment. We make reasonable efforts to protect against computer viruses but we do not accept liability for any liability, loss or damage caused by any computer virus contained in this email. ================================================================================================ From stewjw from gmail.com Tue Aug 4 03:24:45 2009 From: stewjw from gmail.com (StewJW) Date: Tue Aug 4 11:43:49 2009 Subject: Seeking method for fatty acid content determination in plant seeds and seedlings References: Message-ID: <2560f404-c0b6-48c9-bedd-c97e41008456@s15g2000yqs.googlegroups.com> Some commercial lit. can contain some useful info particularly the right type of GC column to use, although I would search the sicientific lit. eg http://www.sge.com/uploads/mF/aa/mFaaBmcFVcEMITZzKHgQmA/BR-0307-C.pdf SGE recommend their BP21- Polyethylene Glycol (TPA treated) or a BPX70-70% Cyanopropyl polysilphenylene-siloxane for the separation of fatty acid methyl esters. There is also a cross-reference guide to other manufacturer columns. Fisher UK who I work for also supply Thermo Hypersil's Trace TR-WAX GC column for FA separation. Make sure you get the right column for the job is the message, its a competative field so you should be able to haggle a good price. From stewjw from gmail.com Tue Aug 4 03:43:02 2009 From: stewjw from gmail.com (StewJW) Date: Tue Aug 4 11:43:53 2009 Subject: Endotoxins References: Message-ID: You could use a 10,000 MWCO ultrafiltration filter. There are a number of manufacturers who produce these depending on the solution volume. A LAL test will determine the endotoxins levels. From stewjw from gmail.com Tue Aug 4 04:00:18 2009 From: stewjw from gmail.com (StewJW) Date: Tue Aug 4 11:43:58 2009 Subject: Endotoxins References: Message-ID: <0964d246-54f7-4d5e-b775-8ba815ab358d@w6g2000yqw.googlegroups.com> Just add typical filter manufacturers who supply ultrafilters are Sartorius, PAL, Millipore, Whatman etc. Another method I know little about is polymyxin B immobilized on Sepharose 4B; I just noticed GE Life Science have removed this from their catalogue, maybe not a popular method. I also came across the review from a Google search, sadly my main source of info these days: http://www.ualberta.ca/~csps/JPPS10_3/MS_996/MS_996.html From agbiok4 from gmail.com Tue Aug 4 07:45:28 2009 From: agbiok4 from gmail.com (kamalaker nasani) Date: Tue Aug 4 11:44:21 2009 Subject: VECTORS SEQUENCES Message-ID: Dear members, Where I can get the sequences of pCAMBIA vectors and other binary vectors and Restriction maps. Is there any free databases available. If any one knows relavent information help me in this. -- Kamalaker Nasani, Biotechnologist, Global Transgenes Ltd. From novalidaddress from nurfuerspam.de Tue Aug 4 12:53:42 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Tue Aug 4 13:19:28 2009 Subject: fusion protein purification problem References: Message-ID: <2c771bb8-bc54-4e69-8217-ee2562050c1f@a13g2000yqc.googlegroups.com> Hi Chris, You might consider immobilizing anti.myc antibodies on an activaed sepharose (eg CNBr, NHS). Should not cross-react with endogenous myc. Wo On 4 Aug., 15:08, Chris McDermott-Roe wrote: > Hi all, > > I have produced a myc-conjugated fusion protein and hope to express it in > mammalian cells and subsequently purify it. ?Are there any myc resins out > there that would permit large-scale purification of my protein? ?Second, if > I was to transfect my protein into say HEK293 cells, would I need to worry > about co-purifying endogenous m-Myc if using such a resin? > > Thanks all > > Chris From engelbert_buxbaum from hotmail.com Tue Aug 4 14:55:09 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Aug 4 16:27:04 2009 Subject: Ab smear obscures signal on IP-western References: Message-ID: Am 03.08.2009, 14:48 Uhr, schrieb DK : > When you denature without reducing conditions, you potentially expose > all those buried cysteines, which are now free to form S-S bridges. When > there are many proteins in the sample, this leads to a mess (particularly > if the samples are heated). The solution is simple: add ~ 50 mM > iodoacetamide > to your SDS page loading buffer (it's soluble in water up to ~ 0.6M). > I do it all the time. Acetamide is not terribly stable and is > light-sensitive, > so use it fresh. NEM is an alternative but IAA reacts faster.r. TCEP (tris-(2-carboxyethyl)-phosphine) is another option, with two caveats: It can not be used in phosphate buffer and it adds charge to the protein, so it is unsuitable before IEF. See Burns et al., J. Org. Chem. 56 (1991) 2648 From hroychow from nmsu.edu Tue Aug 4 19:18:32 2009 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Tue Aug 4 20:02:28 2009 Subject: VECTORS SEQUENCES In-Reply-To: References: Message-ID: <5024.69.95.217.126.1249431512.squirrel@webmail.nmsu.edu> There is this amazing search engine, among several others, where you can simply type in "pcambia" ... We know this search engine as simply "google" If you do not want tot use this very broad serach engine that searches virtually everything in this universe of ours, you may go to the place called "http://www.ncbi.nlm.nih.gov" Type in your query, and go from there. Internet has transformed our lives ... hasn't it? > Dear members, > Where I can get the sequences of pCAMBIA vectors and other binary vectors > and Restriction maps. Is there any free databases available. > If any one knows relavent information help me in this. > > -- > Kamalaker Nasani, > Biotechnologist, > Global Transgenes Ltd. > _______________________________________________ > 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 pattisyang from gmail.com Wed Aug 5 21:40:05 2009 From: pattisyang from gmail.com (Xuan Yang) Date: Thu Aug 6 01:03:59 2009 Subject: Zinc or Iron binding protein, that is a question! In-Reply-To: References: Message-ID: Dear Sir or Madam, The ICP-ES results (detailed in my previous letters) indicated that 1 molar my protein purified from E.coli Origami(DE3) contained about a half molar Zinc and nearly a quarter molar Iron (whether II or III was not available). The protein carried a MBP tag on the N-terminal and the situation was similar with or without His tag at the C terminal. I want to determine whether my protein really bind Zinc or Iron. Does anyone have any experience about such problems? Specifically, now I want to compare the binding efficiency on various IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or CuSO4(control). However, considering the instability of Fe(II) in solution, the design still seemed problematic. Sincerely, Xuan Yang National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, 15 DaTun Road,Chaoyang District, Beijing, China, 100101 Tel: 86-10-64884329 Academic email: YangX@moon.ibp.ac.cn We will either find a way or make one. From virashkgupta from gmail.com Thu Aug 6 06:44:10 2009 From: virashkgupta from gmail.com (Virash Gupta) Date: Thu Aug 6 07:58:45 2009 Subject: Methods Digest, Vol 51, Issue 4-VECTORS SEQUENCES Message-ID: VECTORS SEQUENCES The Cambia vector sequences were distributed free of cost by CAMBIA with necessary software NIT vector to view the same in around 1998. I donot know if they still provide you. If you need I can send you specific sequence, if these could be edited. On 8/4/09, 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. Ab smear obscures signal on IP-western (Lionel Brooks 3rd) > 2. Re: Ab smear obscures signal on IP-western (DK) > 3. fusion protein purification problem (Chris McDermott-Roe) > 4. RNA extraction from blood using Chelex 100 resin? > (Seong Hwan Park) > 5. An effective method for elimination of DNA and RNA residues > (Ovens, Sanja) > 6. Re: Seeking method for fatty acid content determination in > plant seeds and seedlings (StewJW) > 7. Re: Endotoxins (StewJW) > 8. Re: Endotoxins (StewJW) > 9. VECTORS SEQUENCES (kamalaker nasani) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Mon, 03 Aug 2009 12:41:49 -0400 > From: Lionel Brooks 3rd > Subject: Ab smear obscures signal on IP-western > To: methods@magpie.bio.indiana.edu > Message-ID: <4A77134D.7030404@dartmouth.edu> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed > > Hello all, > > I am performing IP-westerns with affinity-purified antibody as the > affinity reagent. > The procedure must be performed under non-reducing conditions. > Unfortunately, when I exclude DTT from my sample loading buffer then all > I can see on the Western is a big Ab smear that runs from ~210 kD to ~20 > kD. > I suspect that the Ab may be partially reduced because there is 1 mM DTT > in my lysis buffer. However I am not sure about this. > Does anyone have any ideas about how I might salvage these samples? > Is there any way to make my Ab stay at the top of the gel? > > thanks, > Lee > > > > ------------------------------ > > Message: 2 > Date: Mon, 03 Aug 2009 18:48:36 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: Ab smear obscures signal on IP-western > To: methods@net.bio.net > Message-ID: > > In article , Lionel > Brooks 3rd wrote: > >Hello all, > > > >I am performing IP-westerns with affinity-purified antibody as the > >affinity reagent. > >The procedure must be performed under non-reducing conditions. > >Unfortunately, when I exclude DTT from my sample loading buffer then all > >I can see on the Western is a big Ab smear that runs from ~210 kD to ~20 > kD. > >I suspect that the Ab may be partially reduced because there is 1 mM DTT > >in my lysis buffer. However I am not sure about this. > >Does anyone have any ideas about how I might salvage these samples? > >Is there any way to make my Ab stay at the top of the gel? > > When you denature without reducing conditions, you potentially expose > all those buried cysteines, which are now free to form S-S bridges. When > there are many proteins in the sample, this leads to a mess (particularly > if the samples are heated). The solution is simple: add ~ 50 mM > iodoacetamide > to your SDS page loading buffer (it's soluble in water up to ~ 0.6M). > I do it all the time. Acetamide is not terribly stable and is > light-sensitive, > so use it fresh. NEM is an alternative but IAA reacts faster. This will > also > "neutralize" all of your DTT in the sample. (Native, non-denatured IgG is > normally not reduced by 1 mM DTT, so it's unlikely that your 20 kDa ~ light > chains are there before you mix your sample with loading buffer. > > DK > > > > > > ------------------------------ > > Message: 3 > Date: Tue, 4 Aug 2009 14:08:45 +0100 > From: Chris McDermott-Roe > Subject: fusion protein purification problem > To: methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Hi all, > > I have produced a myc-conjugated fusion protein and hope to express it in > mammalian cells and subsequently purify it. Are there any myc resins out > there that would permit large-scale purification of my protein? Second, if > I was to transfect my protein into say HEK293 cells, would I need to worry > about co-purifying endogenous m-Myc if using such a resin? > > Thanks all > > Chris > > > ------------------------------ > > Message: 4 > Date: Tue, 4 Aug 2009 11:02:27 +0900 > From: Seong Hwan Park > Subject: RNA extraction from blood using Chelex 100 resin? > To: methods@magpie.bio.indiana.edu > Message-ID: > <7e2b93f90908031902m7461ab77x8211f14c58d2a743@mail.gmail.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Hi, guys. > Does anybody have an experience with RNA extraction from blood using > Chelex 100 resin? > Because I have a DNA version of Chelex 100 resin protocol, I modified it. > > 1. Whole blood 250ul + cell(RBC) lysis buffer 375ul -> 4'C incubation > -> centrifuge -> discard supernatant > 2. repeat 1 twice more > 3. apply 100ul of 5% Chelex 100 resin to the pellet > 4. boiling for 20min > 5. centrifuge and collect supernatant > 6. DNase I treatment for supernatant and heat inactivation of DNase I > 7. RT-PCR > > Do you think this protocol work? > If you have any comment on it, please let me know. > > Seong > > -- > Seong Hwan Park, M.D., Ph.D. > Instructor in Legal Medicine, > College of Medicine, Korea University > 126-1 Anadong 5ga, Seongbukgu, Seoul 136-704 > South Korea > > E-mail: kuforen@gmail.com or pelvis@korea.ac.kr > Tel: 82-2-920-6158 > Fax: 82-2-928-3901 > > > > ------------------------------ > > Message: 5 > Date: Tue, 4 Aug 2009 14:53:53 +1000 > From: "Ovens, Sanja" > Subject: An effective method for elimination of DNA and RNA residues > To: > Message-ID: > > > > Content-Type: text/plain; charset="us-ascii" > > Hi > > Could I have a bit more information on this method? > > Thank you > > Kind regards > > Sanja Ovens > Victoria Police Forensic Services Department > Biology - DNA > 31 Forensic Dve > Macleod > Victoria 3085 > Australia > Email: sanja.ovens@police.vic.gov.au > > > > ================================================================================================ > EMAIL DISCLAIMER > > This email and any attachments are confidential. They may also be subject > to copyright. > > If you are not an intended recipient of this email please immediately > contact us by replying > to this email and then delete this email. > > You must not read, use, copy, retain, forward or disclose this email or any > attachment. > > We do not accept any liability arising from or in connection with > unauthorised use or disclosure > of the information contained in this email or any attachment. > > We make reasonable efforts to protect against computer viruses but we do > not accept liability > for any liability, loss or damage caused by any computer virus contained in > this email. > > ================================================================================================ > > > ------------------------------ > > Message: 6 > Date: Tue, 4 Aug 2009 01:24:45 -0700 (PDT) > From: StewJW > Subject: Re: Seeking method for fatty acid content determination in > plant seeds and seedlings > To: methods@net.bio.net > Message-ID: > <2560f404-c0b6-48c9-bedd-c97e41008456@s15g2000yqs.googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Some commercial lit. can contain some useful info particularly the > right type of GC column to use, although I would search the > sicientific lit. eg > > http://www.sge.com/uploads/mF/aa/mFaaBmcFVcEMITZzKHgQmA/BR-0307-C.pdf > > SGE recommend their BP21- Polyethylene Glycol (TPA treated) or a > BPX70-70% Cyanopropyl polysilphenylene-siloxane for the separation of > fatty acid methyl esters. There is also a cross-reference guide to > other manufacturer columns. Fisher UK who I work for also supply > Thermo Hypersil's Trace TR-WAX GC column for FA separation. Make sure > you get the right column for the job is the message, its a competative > field so you should be able to haggle a good price. > > > ------------------------------ > > Message: 7 > Date: Tue, 4 Aug 2009 01:43:02 -0700 (PDT) > From: StewJW > Subject: Re: Endotoxins > To: methods@net.bio.net > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > You could use a 10,000 MWCO ultrafiltration filter. There are a number > of manufacturers who produce these depending on the solution volume. A > LAL test will determine the endotoxins levels. > > > > ------------------------------ > > Message: 8 > Date: Tue, 4 Aug 2009 02:00:18 -0700 (PDT) > From: StewJW > Subject: Re: Endotoxins > To: methods@net.bio.net > Message-ID: > <0964d246-54f7-4d5e-b775-8ba815ab358d@w6g2000yqw.googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Just add typical filter manufacturers who supply ultrafilters are > Sartorius, PAL, Millipore, Whatman etc. Another method I know little > about is polymyxin B immobilized on Sepharose 4B; I just noticed GE > Life Science have removed this from their catalogue, maybe not a > popular method. I also came across the review from a Google search, > sadly my main source of info these days: > > http://www.ualberta.ca/~csps/JPPS10_3/MS_996/MS_996.html > > > > > ------------------------------ > > Message: 9 > Date: Tue, 4 Aug 2009 18:15:28 +0530 > From: kamalaker nasani > Subject: VECTORS SEQUENCES > To: Plant Tissue Culture , > methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Dear members, > Where I can get the sequences of pCAMBIA vectors and other binary vectors > and Restriction maps. Is there any free databases available. > If any one knows relavent information help me in this. > > -- > Kamalaker Nasani, > Biotechnologist, > Global Transgenes Ltd. > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 51, Issue 4 > ************************************** > -- 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 rominah33 from yahoo.com.ar Thu Aug 6 11:16:56 2009 From: rominah33 from yahoo.com.ar (Romina Alejandra Karas) Date: Thu Aug 6 12:38:03 2009 Subject: I am looking.... Message-ID: <446228.42205.qm@web51502.mail.re2.yahoo.com> Hello, I am looking for anti-mouse CD15 monoclonal antibody (for Flow Cytometry). I am interested in any mAbs to differ murine granulocytes?to MDSC. Romina Alejandra Karas Licenciada en Biotecnolog?a ? ? Yahoo! Cocina Encontra las mejores recetas con Yahoo! Cocina. http://ar.mujer.yahoo.com/cocina/ From cjmcdermottroe from googlemail.com Sun Aug 9 03:19:59 2009 From: cjmcdermottroe from googlemail.com (Chris McDermott-Roe) Date: Sun Aug 9 11:59:29 2009 Subject: fusion protein purification problems Message-ID: Dear all, Many thanks for all the suggestions regarding purification of my myc tagged protein. Chris From mlh_keramati from yahoo.com Mon Aug 10 09:27:24 2009 From: mlh_keramati from yahoo.com (malihe keramati) Date: Mon Aug 10 11:59:26 2009 Subject: pfu pcr Message-ID: <870519.19574.qm@web112519.mail.gq1.yahoo.com> hello all I am trying to pcr out a sequence with pfu. This > has worked before perfectly with taq, but now I get repeatedly emplty lanes. > > I have tried to work with longer extension times as well > as annealing times,gradint pcr and increased enzyme, dnttp's , > primers and mgso4 - but no success - also I digested the > genomic dna (bacterial ) and checked them for pcr . I keep getting > a gel with not the faintest track of a band. -Pfu and its buffer and dntp mix are functional because I check them by positive control and get a sharp band)). - I have checked these temperature :40,45,50,53,56,58,60.for pfu . taq pol pcr has sharp band in range of 52 to 62 C, lower temperature for taq has nonspecific band . the mg conc increased to 3mM and dNTP 0.25 mM of each and pfu conc is 1.25 u/25 microlit . - the segment is about 1300 bp and extension time was set for 3 min in pfu reaction . However in all of experiences there is no pcr product, no band just primer dimmer, Would anyone of you have any idea on that? Best regards, melika . From cathalgarvey from gmail.com Mon Aug 10 12:33:18 2009 From: cathalgarvey from gmail.com (Cathal Garvey) Date: Mon Aug 10 13:09:02 2009 Subject: pfu pcr In-Reply-To: <870519.19574.qm@web112519.mail.gq1.yahoo.com> References: <870519.19574.qm@web112519.mail.gq1.yahoo.com> Message-ID: <468b1a400908101033hf25d58fr23bafd778ce8896c@mail.gmail.com> Hi Malihe, There are a few reasons why you can't get your band using the alternate enzyme. A purely theoretical reason: Depending on the composition of your sequence, a high GC template may be more difficult with an error-checking enzyme, perhaps because the template cannot "mutate" to a more easily amplified form. For example, a knot of high-GC DNA might mutate quickly using Taq, where there is no error checking, because there is a "selective pressure" for template that can be amplified properly. With an error checking enzyme, this is nigh impossible. Leaving that purely theoretical reason aside, it's more likely that pfu's different amplification rate and reaction preferences are to blame. As it is a different enzyme, it can't be assumed it favours the same Mg concentrations etc. Seeing as you've already tried changing all of these things, it could simply be that the extension times are too short or long; taq and pfu amplify at very different rates. Finally, having tried to amplify a horribly complex sequence with an error checking enzyme myself, and having little success worthy of cloning, I would reccomend that you consider having the DNA amplified if you need it only once. If you go over to MrGene.com, they can synthesise the sequence from scratch and send it to you in an ampicillin resistant vector at a competitive price! Just don't trust them to do it as quickly as they advertise if it is a high GC sequence.. 2009/8/10 malihe keramati > > hello all > I am trying to pcr out a sequence with pfu. This > > has worked before perfectly with taq, but now I get repeatedly emplty > lanes. > > > > I have tried to work with longer extension times as well > > as annealing times,gradint pcr and increased enzyme, dnttp's , > > primers and mgso4 - but no success - also I digested the > > genomic dna (bacterial ) and checked them for pcr . I keep getting > > a gel with not the faintest track of a band. > -Pfu and its buffer and dntp mix are functional because I check them by > positive control and get a sharp band)). > - I have checked these temperature :40,45,50,53,56,58,60.for pfu . > taq pol pcr has sharp band in range of 52 to 62 C, lower temperature for > taq has nonspecific band . > the mg conc increased to 3mM and dNTP 0.25 mM of each and pfu conc is 1.25 > u/25 microlit . > - the segment is about 1300 bp and extension time was set for 3 min in > pfu reaction . However in all of experiences there is no pcr product, no > band just primer dimmer, > Would anyone of you have any idea on that? > Best regards, > melika . > > > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- letters.cunningprojects.com twitter.com/onetruecathal Kiva.org - Loans That Change Lives From davidminde from gmail.com Mon Aug 10 12:33:34 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Mon Aug 10 13:09:09 2009 Subject: pfu pcr In-Reply-To: <870519.19574.qm@web112519.mail.gq1.yahoo.com> References: <870519.19574.qm@web112519.mail.gq1.yahoo.com> Message-ID: <123243900908101033o773a88cbje1c33c88a24553a0@mail.gmail.com> ... if your amplicon is "big" (e.g. 5-15 kb), you better choose phusion polymerase (or the like) and it will just work ...(unless your template or primers are degraded or you have problems with primer sequence specificity which can be overcome by prolongation in many cases) best wishes, David 2009/8/10 malihe keramati > > hello all > I am trying to pcr out a sequence with pfu. This > > has worked before perfectly with taq, but now I get repeatedly emplty > lanes. > > > > I have tried to work with longer extension times as well > > as annealing times,gradint pcr and increased enzyme, dnttp's , > > primers and mgso4 - but no success - also I digested the > > genomic dna (bacterial ) and checked them for pcr . I keep getting > > a gel with not the faintest track of a band. > -Pfu and its buffer and dntp mix are functional because I check them by > positive control and get a sharp band)). > - I have checked these temperature :40,45,50,53,56,58,60.for pfu . > taq pol pcr has sharp band in range of 52 to 62 C, lower temperature for > taq has nonspecific band . > the mg conc increased to 3mM and dNTP 0.25 mM of each and pfu conc is 1.25 > u/25 microlit . > - the segment is about 1300 bp and extension time was set for 3 min in > pfu reaction . However in all of experiences there is no pcr product, no > band just primer dimmer, > Would anyone of you have any idea on that? > Best regards, > melika . > > > > > > _______________________________________________ > 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 (magic:) android +31 652614443 home: Griftkade 4 bis 3572 TW Utrecht Science is what happens while we are making other plans (~John Lennon) From pjie2 from cam.ac.uk Mon Aug 10 13:00:56 2009 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Mon Aug 10 15:07:13 2009 Subject: pfu pcr In-Reply-To: References: Message-ID: <7eb5j5F2ffikoU1@mid.individual.net> If it works with Taq, why do you need to use pfu? What can you do with a pfu product that you can't do with a Taq product? Peter From R.Jayakumar from roswellpark.org Mon Aug 10 15:32:36 2009 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Mon Aug 10 17:39:17 2009 Subject: pfu pcr In-Reply-To: <7eb5j5F2ffikoU1@mid.individual.net> References: <7eb5j5F2ffikoU1@mid.individual.net> Message-ID: The existence of 3-5' proof reading exonuclease function and hence lower mispairing might be one reason. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Peter Ellis Sent: Monday, August 10, 2009 2:01 PM To: methods@magpie.bio.indiana.edu Subject: Re: pfu pcr If it works with Taq, why do you need to use pfu? What can you do with a pfu product that you can't do with a Taq product? Peter _______________________________________________ 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 pjie2 from cam.ac.uk Mon Aug 10 16:20:56 2009 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Mon Aug 10 17:39:26 2009 Subject: pfu pcr In-Reply-To: <7eb5j5F2ffikoU1@mid.individual.net> References: <7eb5j5F2ffikoU1@mid.individual.net> Message-ID: <7ebha7F2fagtuU1@mid.individual.net> *sigh* Before I get any more replies telling me "might be a fidelity issue" then yes, I'm aware what pfu is used for. I've also not yet come across an amplicon, particularly one of only 1.3kb, where you couldn't just pick half a dozen clones until you get one with the correct sequence. If there's a particular region that can't amplify properly with Taq (as Cathal Garvey suggests), then that's something the OP needs to tell us about so we can make useful suggestions how to optimise for it. Peter From editor from gene-quantification.info Tue Aug 11 09:12:01 2009 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Tue Aug 11 13:52:54 2009 Subject: qPCR NEWS August 2009 - focus on HRM Message-ID: <7ef73e5b-54ef-42e0-b234-ac7f69047272@a13g2000yqc.googlegroups.com> qPCR NEWS August 2009 - focus on HRM ------------------------------------------------------ Dear researcher, dear Gene Quantification page reader, Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene Quantification homepage. The focus of this newsletter issue is: - Update in HRM papers & HRM dyes =3D> http://HRM.gene-quantification.info - New molecular diagnostics qPCR/real-time PCR discussion forum: LinkedIN & XING - qPCR / real-time PCR BLOG =3D> http://real-time-pcr.blogspot.com/ - For better navigation we developed a TAG CLOUD =3D> http://directory.gene-quantification.info/ - New qPCR events in autumn 2009: symposia & workshops =3D> http://events.gene-quantification.info/ European wide qPCR application workshops =3D> register now ! =3D> course program autumn 2009 & winter 2010 =3D> http://www.gene-quantification.de/bioeps-courses-autumn-2009.pdf ---------------------------------------------------------------------------= ----- Update in HRM papers =3D> http://HRM.gene-quantification.info Determination of KCNQ1OT1 and H19 methylation levels in BWS and SRS patients using methylation-sensitive high-resolution melting analysis. Alders M, Bliek J, vd Lip K, vd Bogaard R, Mannens M. Eur J Hum Genet. 2009 17(4): 467-473. High quality assessment of DNA methylation in archival tissues from colorectal cancer patients using quantitative high-resolution melting analysis. Balic M, Pichler M, Strutz J, Heitzer E, Ausch C, Samonigg H, Cote RJ, Dandachi N. J Mol Diagn. 2009 (2):102-108. Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling. Dobrowolski SF, Gray J, Miller T, Sears M. Hum Mutat. 2009 (6): 891-898. Quantitative evaluation of DNA methylation by optimization of a differential-high resolution melt analysis protocol. Malentacchi F, Forni G, Vinci S, Orlando C. Nucleic Acids Res. 2009 (12): e86 Mutation scanning using high-resolution melting. Taylor CF. Biochem Soc Trans. 2009 37(Pt 2): 433-437. Review. High-resolution melting analysis (HRMA): more than just sequence variant screening. Vossen RH, Aten E, Roos A, den Dunnen JT. Hum Mutat. 2009 30(6): 860-866. Methylation-sensitive high-resolution melting. Wojdacz TK, Dobrovic A, Hansen LL. Nat Protoc. 2008; 3(12): 1903-1908. Primer design versus PCR bias in methylation independent PCR amplifications. Wojdacz TK, Borgbo T, Hansen LL. Epigenetics. 2009 (4): 231-234 Base-pair neutral homozygotes can be discriminated by calibrated high- resolution melting of small amplicons. Gundry CN, Dobrowolski SF, Martin YR, Robbins TC, Nay LM, Boyd N, Coyne T, Wall MD, Wittwer CT, Teng DH. Nucleic Acids Res. 2008 36(10): 3401-3408. High-resolution melting analysis (HRMA):a highly sensitive inexpensive genotyping alternative for population studies. B.L. SMITH, C.-P. LU and J.R. ALVARADO BREMER Molecular Ecology Resources (2009) 1-4 Multiplex Amplicon Genotyping by High-Resolution Melting. Michael T.Seipp, Jacob D. Durtschi, Karl V. Voelkerding, and Carl T. Wittwer Communication in Journal of Biomolecular Techniques (2009) 20: 160=96164 ... and much more =3D> http://HRM.gene-quantification.info ---------------------------------------------------------------------------= ----- New HRM & qPCR Dye - Chromofy Chromofy is a monomeric asymmetric cyanine dye, developed by TATAA Biocenter for use in qPCR applications. The dye has absorbance and emission wavelengths that are suitable for the FAM/SYBR channel on most common real-time PCR instruments. When binding to dsDNA Chromofy shows a very strong fluorescence increase download flyer =3D> http://www.gene-quantification.de/chromofy-flyer.pdf Chromofy in High-resolution melting High-resolution melting is a post-PCR analysis that discriminates different samples on the basis of their melting temperature. Chromofy and HRM can be used for genotyping, either with ro without unlabelled probes and for methylation analysis. Methylation analysis is performed after bisulfite treatment of the DNA samples which converts unmethylated cytosines to uracils while methylated cytosines remain intact. During subsequent PCR the uracils will be substituted for thymines giving rise to a Tm difference between methylated and un- methylated samples, which is seen as varying heights of the plateu in the normalized HRM data. Using Chromofy your assay can be sensitive down to 1 % methylated DNA in un-methylated background. Download Chromofy user manual =3D> http://www.gene-quantification.de/chromo= fy-flyer.pdf ---------------------------------------------------------------------------= ----- 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 2010 in Vienna International qPCR 2010 symposium in Vienna 7th =96 9th April 2010 The focus of the qPCR 2010 Event will be =93The ongoing evolution of qPCR=94 representing all new and emerging techniques, applications and data analysis methods. HRM, microRNA, CNV, single-cell qPCR, digital PCR, and analysis of circulating nucleic acids will be in the focus of the conference. http://www.qpcr2010-vienna.net/ download first announcement =3D> http://www.bioeps.com/qpcr2010/Vienna-qPCR= -2010-1st-announcement.pdf ---------------------------------------------------------------------------= ----- 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.) 14 - 16 September 2009 (E) NEW single-cell qPCR 19 - 23 September 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2-day BioStatistics (Thu. - Fri.) 26 - 28 October 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) 16 - 20 November 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2-day BioStatistics (Thu. - Fri.) 7 - 11 December 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics (Thu. - Fri.) =3D> http://www.gene-quantification.de/bioeps-courses-autumn-2009.pdf ---------------------------------------------------------------------------= ----- Forward Please send the qPCR NEWS to further scientists and friends who are interested in qPCR ! Best regards, Michael W. Pfaffl responsible Editor of the Gene Quantification Pages http://www.gene-quantification.info ---------------------------------------------------------------------------= ----- If this newsletter is not displayed correctly by your email client, please use following link: http://qpcrnews.gene-quantification.info/ The qPCR NEWS and the Gene Quantification Pages are educational sites with the only purpose of facilitating access to qPCR related information on the internet. The qPCR NEWS and the Gene Quantification Pages are edited by Michael W. Pfaffl. Copyright =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 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 virashkgupta from gmail.com Mon Aug 10 23:37:27 2009 From: virashkgupta from gmail.com (Virash Gupta) Date: Tue Aug 11 13:52:59 2009 Subject: Methods Digest, Vol 51, Issue 9 In-Reply-To: <200908101705.n7AH5cp08669@net.bio.net> References: <200908101705.n7AH5cp08669@net.bio.net> Message-ID: Check with normal taq for integrity of template DNA On 8/10/09, 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. pfu pcr (malihe keramati) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Mon, 10 Aug 2009 07:27:24 -0700 (PDT) > From: malihe keramati > Subject: pfu pcr > To: "methods@iubio.bio.indiana.edu" > Message-ID: <870519.19574.qm@web112519.mail.gq1.yahoo.com> > Content-Type: text/plain; charset=us-ascii > > > hello all > I am trying to pcr out a sequence with pfu. This > > has worked before perfectly with taq, but now I get repeatedly emplty > lanes. > > > > I have tried to work with longer extension times as well > > as annealing times,gradint pcr and increased enzyme, dnttp's , > > primers and mgso4 - but no success - also I digested the > > genomic dna (bacterial ) and checked them for pcr . I keep getting > > a gel with not the faintest track of a band. > -Pfu and its buffer and dntp mix are functional because I check them by > positive control and get a sharp band)). > - I have checked these temperature :40,45,50,53,56,58,60.for pfu . > taq pol pcr has sharp band in range of 52 to 62 C, lower temperature for > taq has nonspecific band . > the mg conc increased to 3mM and dNTP 0.25 mM of each and pfu conc is 1.25 > u/25 microlit . > - the segment is about 1300 bp and extension time was set for 3 min in > pfu reaction . However in all of experiences there is no pcr product, no > band just primer dimmer, > Would anyone of you have any idea on that? > Best regards, > melika . > > > > > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 51, Issue 9 > ************************************** > -- 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 Isha.Patel from fda.hhs.gov Tue Aug 11 14:16:49 2009 From: Isha.Patel from fda.hhs.gov (Patel, Isha*) Date: Tue Aug 11 15:20:35 2009 Subject: DNA purification after agarase treatment Message-ID: <73DC693AEF5AB944B788A146B20CE14502043A82@FMD3VS012.fda.gov> Hi, Did you end up doing the drop dialysis or precipitation? I have a large 100kb plasmid and don't know what to do as I don't want to lose a lot. I want to use the plasmid for cloning and sequencing after extraction. Thanks in advance for your input, Isha Patel U.S.FDA, OARSA, CFSAN, DMB, 8302 Muirkirk Rd, Room 3416, Laurel, MD 20708 isha.patel@fda.hhs.gov From jburdach from gmail.com Tue Aug 11 16:47:31 2009 From: jburdach from gmail.com (Jon Burdach) Date: Tue Aug 11 20:06:55 2009 Subject: pfu pc Message-ID: <2ae8ce280908111447i34a448fag90741845e0c7ac7a@mail.gmail.com> Try adding 10% DMSO to your PCR tube. It helps prevent secondary structure formation for G-C rich amplicons. I was trying to get a pfu PCR working for ages and it worked perfectly as soon as I added DMSO. Jon From rahimpour_a from yahoo.com Fri Aug 14 03:07:55 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Fri Aug 14 10:49:18 2009 Subject: seap reporter vector Message-ID: <883202.86112.qm@web52712.mail.re2.yahoo.com> Dear All Hi I am looking for some mammalian?Secretary reporter genes like SEAP, I know there are some commercial suppliers but unfortunately I am not able to purchase any. is there anybody that can kindly provide that reporter construct to me? ? Best Wishes Azam From peter.ianakiev from gmail.com Fri Aug 14 09:24:29 2009 From: peter.ianakiev from gmail.com (peter) Date: Fri Aug 14 10:49:38 2009 Subject: Silver/gold staining and PCR amplification Message-ID: Hi Fellows Researchers, I have a question and someone might have an answer - can DNA that has been silver or nonogold stained be used in amplification or hibridization? Thanks Peter From hiraghuraman from gmail.com Sun Aug 16 13:33:37 2009 From: hiraghuraman from gmail.com (Raghu) Date: Sun Aug 16 15:12:25 2009 Subject: miRNA target Message-ID: <18ed2061-bf08-413e-a0a4-429de65cf68c@o15g2000yqm.googlegroups.com> Dear all. I have a list of miRNAs in Oryza sativa and I am looking for their target mRNAs. I am not able to get any software that will search the targets in O.sativa. Please help me if anyone knows it. Thank you... Raghu Ram B From forumfede from gmail.com Tue Aug 18 13:10:58 2009 From: forumfede from gmail.com (Fede Curzio) Date: Tue Aug 18 18:00:48 2009 Subject: Solvent Evaporation suggestions Message-ID: <3c532d20908181110j535b1321sf189bcdc3675ffa9@mail.gmail.com> Dear Members, thanks in advance to you all for any help I can receive. I have this chemical, named 4-hydroxy nonenal that comes around 65 mM in 100% ethanol. I need to concentrate it at list to 200 mM (it is still soluble in ethanol). So data sheet suggest to evaporate the solvent under Nitrogen stream at room temperature and then dissolve again the remained compund. I do not like too much this procedure because this compound it is not very stable at room temperature and in contact with air. Do anybody have some suggestions for a different procedure ? Thanks Federico From gkapolas from yahoo.gr Wed Aug 19 05:15:33 2009 From: gkapolas from yahoo.gr (george kapolas) Date: Wed Aug 19 10:00:44 2009 Subject: VECTORS SEQUENCES Message-ID: <189435.28376.qm@web25008.mail.ukl.yahoo.com> All informations about pCambia vectors are proviided to the following site: www.cambia.org ___________________________________________________________ ?????????????? Yahoo!; ?????????? ?? ?????????? ???????? (spam); ?? Yahoo! Mail ???????? ??? ???????? ?????? ????????? ???? ??? ??????????? ????????? http://login.yahoo.com/config/mail?.intl=gr From rahimpour_a from yahoo.com Sat Aug 22 04:37:38 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Sat Aug 22 12:55:33 2009 Subject: copy number determination real time vs sauthern blott Message-ID: <434937.50518.qm@web52705.mail.re2.yahoo.com> Dear All I am going to determine copy number of integrated genes in transfected cells, most articles used sauthern blotting for this purpose. Is it possible to use quantitative real time pcr instead of that? and is it as accurate as sauthern blotting in identification of single copies? best From martinseac from gmail.com Sat Aug 22 15:44:57 2009 From: martinseac from gmail.com (Emerson A. Castilho Martins) Date: Sat Aug 22 21:35:38 2009 Subject: copy number determination real time vs sauthern blott In-Reply-To: <434937.50518.qm@web52705.mail.re2.yahoo.com> References: <434937.50518.qm@web52705.mail.re2.yahoo.com> Message-ID: <1250973897.4761.3.camel@emerson-laptop> In fact, Real Time PCR is much more sensitive than southern blot, but you have to use standard curve for determination of this. Em S?b, 2009-08-22 ?s 02:37 -0700, Azam Rahimpour escreveu: > Dear All > I am going to determine copy number of integrated genes in transfected cells, most articles used sauthern blotting for this purpose. Is it possible to use quantitative real time pcr instead of that? and is it as accurate as sauthern blotting in identification of single copies? > best > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods From shifalich from rediffmail.com Sun Aug 23 03:01:59 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Sun Aug 23 12:40:35 2009 Subject: Redissolving lyophilised proteins? Message-ID: <1249409953.S.3691.H.TldTAFJlOiBmdXNpb24gcHJvdGVpbiBwdXJpZmljYXRpb24gcHJvYmxlbQ__.R.rfs26, rfs26, R, 2499, 19693.63829.f4mail-235-209.rediffmail.com.old.1251014519.61031@webmail.rediffmail.com> Dear all! I am expressing my protein in Origami Strain transformed with pET 32a containing my protein of interest. The protein is expressed as a fusion protein with Thioredoxin tag and His tag with enterokinase (Ek)cleavage site. After purification on Ni-NTA column, I purify the recombinant protein on Reverse phase HPLC and eluted protein is lyophilised. Then, I try to dissolve the protein in Ek digestion buffer but the powder does not go into solution completely even dissolving the protein in appropriate buffer and pH. I loose almost 80-90% ptotrin there. Even soncation doesn't help protein dissolution. Can anybody suggest how do I dissolve the protein powder completely? Second problem is: After digestion, protein is again purified on C18 column (It is 10kDa protein). Protein gets eluted in multiple pks. My protein has 10 Cys residues and is expected to form 5 disulphide bonds. Are these multiple pks. because of isoforms formed because of disuphide pairing? Due to this the yields drops down to few micrograms/l of bacterial culture. How do I overcome this problem? I am already using Origami strain. Do I again have to denature, refold and purify thr protein again? Does anybody have a protocol that they have tried and works for such small proteins with so many disuphide bonds? Please share with me. Please send your responses even if you can help in soving even a single problem out of all above. Your kind help will be highly appreciated. Thanks Regards Shifali On Tue, 04 Aug 2009 23:49:13 +0530 wrote >Hi Chris, You might consider immobilizing anti.myc antibodies on an activaed sepharose (eg CNBr, NHS). Should not cross-react with endogenous myc. Wo On 4 Aug., 15:08, Chris McDermott-Roe wrote: > Hi all, > > I have produced a myc-conjugated fusion protein and hope to express it in > mammalian cells and subsequently purify it. Are there any myc resins out > there that would permit large-scale purification of my protein? Second, if > I was to transfect my protein into say HEK293 cells, would I need to worry > about co-purifying endogenous m-Myc if using such a resin? > > Thanks all > > Chris _______________________________________________ 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 bzahedi from bccrc.ca Sun Aug 23 16:37:52 2009 From: bzahedi from bccrc.ca (Bari Zahedi) Date: Sun Aug 23 18:11:32 2009 Subject: low LPS plasmid prep protocol Message-ID: Does anyone have a non-kit plasmid prep protocol for cell transfection that will generate plasmid with low LPS levels? I will be using the plasmids to transfect immune cells. Thanks! From nick.theodorakis from gmail.com Mon Aug 24 12:32:16 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Mon Aug 24 15:03:17 2009 Subject: low LPS plasmid prep protocol References: Message-ID: On Aug 23, 4:37?pm, Bari Zahedi wrote: > Does anyone have a non-kit plasmid prep protocol for cell transfection that will generate plasmid with low LPS levels? I will be using the plasmids ?to transfect immune cells. Thanks! Doing a double-banding in CsCl after alkaline lysis should be pretty low in LPS, but I doubt that's much cheaper than using a commercial kit, and you need access to an ultracentrifuge and the appropriate rotor. And it's a big pain. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From aawara from pontiff-playground.org Mon Aug 24 18:34:15 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Mon Aug 24 21:30:20 2009 Subject: low LPS plasmid prep protocol References: Message-ID: In , Nick Theodorakis wrote: > On Aug 23, 4:37?pm, Bari Zahedi wrote: >> Does anyone have a non-kit plasmid prep protocol for cell transfection that will generate plasmid with low LPS levels? I will be using the plasmids ?to transfect immune cells. Thanks! > > Doing a double-banding in CsCl after alkaline lysis should be pretty > low in LPS, but I doubt that's much cheaper than using a commercial > kit, and you need access to an ultracentrifuge and the appropriate > rotor. This is how my lab does it, using a VTi90 rotor that cost an arm and a leg. But we get both spins done in one day - 4 hours spins at 70K. After the second spin, it takes us about 6 extractions with saturated butanol to get the ethidium bromide out. We precipitate the DNA out of CsCl using ethanol, rather than an overnight dialysis. Typically from a 400 ml culture (in terrific broth), we recover about 300 - 600 micrograms of pBR322-based low copy plasmids. It is, as you say, a pain. But on the other hand, we get excellent transfections of primary cells (lymphocytes in our case) using nucleofactor electroporations. -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From aawara from pontiff-playground.org Mon Aug 24 20:50:10 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Mon Aug 24 21:30:27 2009 Subject: low LPS plasmid prep protocol References: Message-ID: In , DK wrote: > Wow, so much work for so little plasmid. Just out of curiosity: > why low copy plasmids for mammalian transfections? Highly > repetitive sequences or what? One would think that simply > by swapping an ori you'd get your purity up ~10X. Many repeats. And all that work is worth it for the reproducibility. > Not to be an asshole, but I have to ask: Aawara, do you guys > always do empty vector transfection controls? All the time. I didn't know that all nucleofactor had was a peptide - that's worth trying. Many years ago I was told that some of the nucleofactor buffers contain a low level of wortmannin. The idea being that transfected DNA contains nicks etc. that activate ATM - possibly resulting in apoptosis of transfected cells. The low level of wortmannin apparently attenuates ATM activation, while not completely blocking repair - permitting transfected cells time to repair DNA and survive. -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From nick.theodorakis from gmail.com Mon Aug 24 21:13:14 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Mon Aug 24 21:30:35 2009 Subject: low LPS plasmid prep protocol References: Message-ID: <767ef193-ca21-4e11-8ef2-3b858f67f46e@b14g2000yqd.googlegroups.com> On Aug 24, 8:50?pm, Aawara Chowdhury wrote: [...] > > I didn't know that all nucleofactor had was a peptide - that's > worth trying. ?Many years ago I was told that some of the nucleofactor > buffers contain a low level of wortmannin. ... Wow, seriously? And they didn't disclose that? You'd think people would want to know something like that. High copy plasmids are great when you can use them. I used to max out the Qiagen Maxi preps (> 500 ug) from 50 ml superbroth culture using Bluescript-derived vectors, and even more yield from a CsCl prep back when I did them (we could often pull the band just viewing under room lighting). > The idea being that transfected DNA contains nicks etc. that activate ATM - possibly > resulting in apoptosis of transfected cells. It seems in that case then one advantage of using CsCl is that one can grab only the supercoiled form and forget the nicked version. I thought I remembered reading that nicked forms are more prevalent when using chloramphenicol amplification (as one might when using pBR). Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From bzahedi from bccrc.ca Tue Aug 25 02:11:29 2009 From: bzahedi from bccrc.ca (Bari Zahedi) Date: Tue Aug 25 13:20:51 2009 Subject: low LPS plasmid prep protocol In-Reply-To: References: , Message-ID: Thanks all for your answers. First, appologies, i just sent an email asking Nick something i wanted to ask aawara. I have some follow up questions for you guys. First, i have too many constructs and too many transfections to be able to do CsCl plasmid purification. Any of you tried PEG ppt'n after alkali lysis and used the DNA for electroporation or nucleofection? We are not set up for CsCl purifications but used to routinely do PEG ppt'ns in the lab for sequencing back in the day. Next, aawara, do you ever transfect GFP-tagged constructs into primary lymphocytes?If so, what was the transfection efficiency- ie % +ve cells? i would like to transfect primary murine splenic B cells using nucleofection. DK, i'm very intrigued with your suggestion to increase electroporation efficiency with histones. The only reason i am trying nucleofection is because i get really low transfection of my cell line. I mostly first, am interested in increasing my electroporation efficiency, second, in expressing various constructs transiently in various lymphocyte cell lines. Any advice would be appreciated on how to increase my electroporation efficiency. Thanks all! "Nucleofector and nucleofector is a marketing device. There is nothing new or original in it. It's just a simple electroporator, pretty generic HEPES-based buffer and an alkaline peptide that is efficiently targeted to the nucleus. In Amaxa's case, it is most likely MEEDTPPIKKKRKVEDL at 25 microM final, a neutral charge fusion of NLS of SV40 large T-antigen and N-terminal sequence of VP2 protein. The use of DNA-binding peptides (and this one, specifically) to enhance transfection was reported and patented at least 10 and 5 years before the Amaxa's patent, respectively. So Amaxa's patent is just a scam to circumvent existing patents, and the "revolutionary technology" for which absolutely no hint is available throught the company is nothing by a marketing ploy. Not to be an asshole, but I have to ask: Aawara, do you guys always do empty vector transfection controls? In more than one papers I saw "nucleofection" used without such control. How do I know the effects were not due to the magic bufferyou observe after transfections relate to peptide's presense? DK P.S. I have spiked plasmid with histones from Sigma to observe increase of electroporation efficiency 20 years ago; 15 years ago the same was repeated in the lab accross the hall in Paramecium electroporations; neither was ever published, though)." ________________________________________ From: methods-bounces@oat.bio.indiana.edu [methods-bounces@oat.bio.indiana.edu] On Behalf Of DK [dk@no.email.thankstospam.net] Sent: August 24, 2009 7:20 PM To: methods@magpie.bio.indiana.edu Subject: Re: low LPS plasmid prep protocol In article , aawara@pontiff-playground.org wrote: >All the time Good to hear! I hope it means "every time" :-))) >I didn't know that all nucleofactor had was a peptide - that's >worth trying. For a 100% reliable identification, give a small sample to a reliable mass-spec facility, asking to look for peptides. That it is a peptide I have no doubt. The only question is which one of the listed is being used. >Many years ago I was told that some of the nucleofactor >buffers contain a low level of wortmannin. The idea being that >transfected DNA contains nicks etc. that activate ATM - possibly >resulting in apoptosis of transfected cells. The low level of >wortmannin apparently attenuates ATM activation, while not completely >blocking repair - permitting transfected cells time to repair DNA >and survive. Doubt it very much. Certainly the patent application (US 2008/0145938) makes no mention of it. And certainly no evidence was ever presented that it is viability enhancement that is a key to the claimed black magic of nucleofection. My reading of the way the patent is written is that it mostly has this bogus "high buffer"/"maybe, just maybe high salt" thing (which is pretty generic anywau and is super unlikely to be crucial) to silently slip in peptides, which is absolutely a rip off of others' ideas. DK _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods ________________________________________ From: methods-bounces@oat.bio.indiana.edu [methods-bounces@oat.bio.indiana.edu] On Behalf Of DK [dk@no.email.thankstospam.net] Sent: August 24, 2009 7:20 PM To: methods@magpie.bio.indiana.edu Subject: Re: low LPS plasmid prep protocol In article , aawara@pontiff-playground.org wrote: >All the time Good to hear! I hope it means "every time" :-))) >I didn't know that all nucleofactor had was a peptide - that's >worth trying. For a 100% reliable identification, give a small sample to a reliable mass-spec facility, asking to look for peptides. That it is a peptide I have no doubt. The only question is which one of the listed is being used. >Many years ago I was told that some of the nucleofactor >buffers contain a low level of wortmannin. The idea being that >transfected DNA contains nicks etc. that activate ATM - possibly >resulting in apoptosis of transfected cells. The low level of >wortmannin apparently attenuates ATM activation, while not completely >blocking repair - permitting transfected cells time to repair DNA >and survive. Doubt it very much. Certainly the patent application (US 2008/0145938) makes no mention of it. And certainly no evidence was ever presented that it is viability enhancement that is a key to the claimed black magic of nucleofection. My reading of the way the patent is written is that it mostly has this bogus "high buffer"/"maybe, just maybe high salt" thing (which is pretty generic anywau and is super unlikely to be crucial) to silently slip in peptides, which is absolutely a rip off of others' ideas. DK _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From engelbert_buxbaum from hotmail.com Tue Aug 25 08:58:41 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Aug 25 13:21:09 2009 Subject: Redissolving lyophilised proteins? References: Message-ID: Am 23.08.2009, 04:01 Uhr, schrieb shifali chatrath : > After purification on Ni-NTA column, I purify the recombinant protein on > Reverse phase HPLC and eluted protein is lyophilised. Then, I try to > dissolve the protein in Ek digestion buffer but the powder does not go > into solution completely even dissolving the protein in appropriate > buffer and pH. I loose almost 80-90% ptotrin there. I am not surprised. RPC can be used for peptides, but for proteins it usually results in loss of activity and aggregation. I would use a more gentle method, and keep the protein in solution at all times. Since you got considerable pre-purification by affinity chromatography, which also results in a very concentrated product, you may get away with polishing by gel filtration on a high res medium. During this step, you can at the same time exchange the buffer against what ever you need for the next step. > After digestion, protein is again purified on C18 column (It is 10kDa > protein). Protein gets eluted in multiple pks. My protein has 10 Cys > residues and is expected to form 5 disulphide bonds. That's a case of GIGO (garbage in, garbage out). Digestion will be incomplete on such an aggregate. If a homogeneous product, resulting from proper purification, still gives problems with digestion, you can try to completely reduce the protein and then try again. From engelbert_buxbaum from hotmail.com Tue Aug 25 09:20:31 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Aug 25 13:21:19 2009 Subject: low LPS plasmid prep protocol References: Message-ID: Am 24.08.2009, 20:49 Uhr, schrieb DK : > Another thought on LPS removal: LPS has lipid and DNA > does not. LPS must bind to resins designed for lipid removal. > Bio-Rad, Calbiochem and Pierce sell them. (The > polymixin-based sorbents are more selective, way more > expensive and are designed to not bind proteins so much and > detergent removal ones do; so for DNA purification simple > and quite cheap detergent binding resins should do fine). I have never worked with DNA, but should ethanol precipitation not remove most LPS with the supernatant? Btw, the detergent removal gels, when used properly, do not bind much protein. Rigaud's group has done extensive work on that, for a review see @article{Rig-98, AUTHOR= {J.L. Rigaud}, TITLE= {Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-{D} crystals}, YEAR= {2002}, JOURNAL= {Braz. J. Med. Biol. Res.}, PAGES= {753-766}, VOLUME= {35}, DOI= {10.1590/S0100-879X2002000700001}, URL= {http://www.scielo.br/pdf/bjmbr/v35n7/4512.pdf}, LANGUAGE= {engl} } From nick.theodorakis from gmail.com Tue Aug 25 09:52:32 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Tue Aug 25 13:21:25 2009 Subject: low LPS plasmid prep protocol References: Message-ID: On Aug 25, 9:20?am, "Dr Engelbert Buxbaum" wrote: > Am 24.08.2009, 20:49 Uhr, schrieb DK : > > > Another thought on LPS removal: LPS has lipid and DNA > > does not. LPS must bind to resins designed for lipid removal. > > Bio-Rad, Calbiochem and Pierce sell them. (The > > polymixin-based sorbents are more selective, way more > > expensive and are designed to not bind proteins so much and > > detergent removal ones do; so for DNA purification simple > > and quite cheap detergent binding resins should do fine). > > I have never worked with DNA, but should ethanol precipitation not remove ? > most LPS with the supernatant? It does not. See: for example: 1.3.3.1 Rapid Isolation Micro Method for LPS (20) 1. Centrifuge a bacterial suspension (108 colony-forming units/mL in 2 mL PBS) in a 15-mL tube at 10,000?g for 5?min. Wash the pellet once in PBS (pH 7.2) containing 0.15?mM CaCl2 and 0.5?mM MgCl2. Resuspend washed cells in 300?L of water and transfer to a 1-dram vial containing a stir bar. 2. Add an equal volume of hot (65?70?C) 90% phenol. Stir the mixture vigorously at 65?70? for 15?min. Chill suspension on ice, transfer to a 1.5-mL polypropylene tube, and centrifuge at 8500?g for 15?min. 3. Transfer supernate to a 15-mL conical centrifuge tube. Re-extract phenol phase with 300??L of distilled water. Pool the aqueous phases. 4. Add sodium acetate to 0.5 M final concentration. Add 10 volume of 95% ethanol and place sample at ?20?C overnight in order to precipitate the LPS. Centrifuge precipitate at 2000?g at 4?C for 10 min. Discard supernatant. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From lu1m from cmich.edu Tue Aug 25 14:19:41 2009 From: lu1m from cmich.edu (Lu, Ming ) Date: Tue Aug 25 15:46:02 2009 Subject: DNA isolation from hair Message-ID: <15688503C68EED4C81F9E79C4FABA19102EF41B0@cmail7.central.cmich.local> Gary: Could you send me a protocol for the DNA isolation? Thanks! Ming Lu From aawara from pontiff-playground.org Tue Aug 25 20:37:29 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Wed Aug 26 10:55:25 2009 Subject: low LPS plasmid prep protocol References: Message-ID: In , Bari Zahedi wrote: > Next, aawara, do you ever transfect GFP-tagged constructs into primary lymphocytes?If so, what was the transfection efficiency- ie % +ve cells? i would like to transfect primary murine splenic B cells using nucleofection. Primary human B-cells that are stimulated with pokeweed mitogen. With an Amaxa, we can get about 20% - 30% transfection, with no detectable apoptosis. We've been unable to transfect them using any other method. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From Craig.McAndrew from icr.ac.uk Wed Aug 26 04:46:12 2009 From: Craig.McAndrew from icr.ac.uk (Craig McAndrew) Date: Wed Aug 26 10:55:32 2009 Subject: Semi sticky surface for shaking platform Message-ID: <20090826T104612Z_CBFB000F0000@icr.ac.uk> Hi all, in the distant past the lab I was in obtained a blue semi sticky matt that you could use on shaking platforms. I say semi sticky as the matt would lift off the platform a little when you removed the flasks. It held flasks on at 140 rpm but wasn't adhered to the platform. Has anyone used anything similar and if so do you know where you got it from. Thanks Craig The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network. From sanchayita_kar from yahoo.com Wed Aug 26 00:19:23 2009 From: sanchayita_kar from yahoo.com (Sanchayita Kar) Date: Wed Aug 26 11:05:21 2009 Subject: Share Research Reagents Message-ID: <949178.76841.qm@web50009.mail.re2.yahoo.com> Sciclips has created a unique platform where you can share or sell research reagents prepared in your laboratory. These reagents can be antibodies, cell lines, vectors, cDNAs, small molecules, recombinant proteins, oligonucleotides, peptides, research kits?etc. Please visit www.sciclips.com for more details. --- From silverstein.joshua from gmail.com Wed Aug 26 11:18:45 2009 From: silverstein.joshua from gmail.com (Joshua Silverstein) Date: Wed Aug 26 12:30:45 2009 Subject: Semi sticky surface for shaking platform In-Reply-To: <20090826T104612Z_CBFB000F0000@icr.ac.uk> References: <20090826T104612Z_CBFB000F0000@icr.ac.uk> Message-ID: <13934d290908260918h750220c6u480c029699ace7bd@mail.gmail.com> I believe that you can get something like that from a Bed Bath and Beyond store (I don't know if they have those in the UK). Basically, they have sticky pads that you can put under a throw rug in the house... They are very similar to the material you are referring to. Here's a link to what I am talking about: http://reviews.overstock.com/9876/376326/reviews.htm. Hope this helps. Josh On Wed, Aug 26, 2009 at 5:46 AM, Craig McAndrew wrote: > Hi all, in the distant past the lab I was in obtained a blue semi sticky > matt that you could use on shaking platforms. I say semi sticky as the matt > would lift off the platform a little when you removed the flasks. It held > flasks on at 140 rpm but wasn't adhered to the platform. > > Has anyone used anything similar and if so do you know where you got it > from. > > Thanks > > Craig > > The Institute of Cancer Research: Royal Cancer Hospital, a charitable > Company Limited by Guarantee, Registered in England under Company No. 534147 > with its Registered Office at 123 Old Brompton Road, London SW7 3RP. > > This e-mail message is confidential and for use by the addressee only. If > the message is received by anyone other than the addressee, please return > the message to the sender by replying to it and then delete the message from > your computer and network. > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From davidminde from gmail.com Wed Aug 26 11:26:25 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Wed Aug 26 12:30:51 2009 Subject: Semi sticky surface for shaking platform In-Reply-To: <20090826T104612Z_CBFB000F0000@icr.ac.uk> References: <20090826T104612Z_CBFB000F0000@icr.ac.uk> Message-ID: <123243900908260926g1e609c41rf98099b63b0d178e@mail.gmail.com> Infors has nice ''sticky stuff". That one is really sticky though ... and green ;) http://www.infors-ht.com It is less effective with plastic than glass surfaces. (E.g. a beverage 2L bottle will fly away @ 200 rpm) but app. fine with multiwell plates for e.g. insect-cell screens. best wishes, David 2009/8/26 Craig McAndrew > Hi all, in the distant past the lab I was in obtained a blue semi sticky > matt that you could use on shaking platforms. I say semi sticky as the matt > would lift off the platform a little when you removed the flasks. It held > flasks on at 140 rpm but wasn't adhered to the platform. > > Has anyone used anything similar and if so do you know where you got it > from. > > Thanks > > Craig > > The Institute of Cancer Research: Royal Cancer Hospital, a charitable > Company Limited by Guarantee, Registered in England under Company No. 534147 > with its Registered Office at 123 Old Brompton Road, London SW7 3RP. > > This e-mail message is confidential and for use by the addressee only. If > the message is received by anyone other than the addressee, please return > the message to the sender by replying to it and then delete the message from > your computer and network. > > _______________________________________________ > 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 Science is what happens while we are making other plans (~John Lennon)