Methods Digest, Vol 52, Issue 7

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Fri Sep 11 22:23:45 EST 2009


Besides all other answers, try this- If plasmid is there it will be
isolated. If you using old solutions or old kit for plasmid purification-
the main culprit is solution 2- alkaline lysis solution. It contains NaOH
and as it gets repeatedel exposed to air-it absorbs carbon dioxide and
converted to sod carbonate- to result in decreased alkalinity. The
solution becomes inefficient in appropriate lysis. So prepare fresh solution
and use.

On 9/11/09, methods-request from oat.bio.indiana.edu <
methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
>
>   1. pTopo purification problems (Yoram Gerchman)
>   2. Re: pTopo purification problems
>      (Taliaferro, Dwayne (NIH/NIMH) [F])
>   3. RE: pTopo purification problems (Bari Zahedi)
>   4. Re: pTopo purification problems (Duncan Clark)
>   5. Re: pTopo purification problems (Cathal Garvey)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Thu, 10 Sep 2009 22:54:58 +0300
> From: Yoram Gerchman <gerchman from research.haifa.ac.il>
> Subject: pTopo purification problems
> To: methods from oat.bio.indiana.edu
> Message-ID: <1252612498.4aa95992c8c3a from webmail.haifa.ac.il>
> Content-Type: text/plain; charset=windows-1255
>
>
> Greetings netters
> I have recently made an attempt to clone a short gene into pTopo (using
> Top10
> competent cells). i did get white colonies on LB-Amp-X-gal, and they grew
> nicely on liquid LB-Amp, but after plasmid purification I get no DNA (tried
> already 2 different kits). Any ideas as to how remediate this situation?
> Many thanks
> Yoram
>
>
>
> ------------------------------------------------------------------------
> This message was sent using IMP, the Webmail Program of Haifa University
>
>
>
> ------------------------------
>
> Message: 2
> Date: Thu, 10 Sep 2009 17:31:20 -0400
> From: "Taliaferro, Dwayne (NIH/NIMH) [F]" <taliaferrod from mail.nih.gov>
> Subject: Re: pTopo purification problems
> To: "methods from oat.bio.indiana.edu" <methods from oat.bio.indiana.edu>
> Message-ID: <C6CEE868.4DD0%taliaferroD from mail.nih.gov<C6CEE868.4DD0%25taliaferroD from mail.nih.gov>
> >
> Content-Type: text/plain; charset="iso-8859-1"
>
> Um...try it again?
>
> On a constructive note...
>
> What size is the gene?
> Did you -tail it?
> Did you try a control reaction?
> Perhaps it was a plasmid prep issue?
> Are you sure the colony you picked was bacteria vs fungus?
> Are you sure your AMP is working?
>
> On 9/10/09 3:54 PM, "Yoram Gerchman" <gerchman from research.haifa.ac.il>
> wrote:
>
>
>
> Greetings netters
> I have recently made an attempt to clone a short gene into pTopo (using
> Top10
> competent cells). i did get white colonies on LB-Amp-X-gal, and they grew
> nicely on liquid LB-Amp, but after plasmid purification I get no DNA (tried
> already 2 different kits). Any ideas as to how remediate this situation?
> Many thanks
> Yoram
>
>
>
> ------------------------------------------------------------------------
> This message was sent using IMP, the Webmail Program of Haifa University
>
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> Methods mailing list
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>
> ------------------------------
>
> Message: 3
> Date: Thu, 10 Sep 2009 16:48:19 -0700
> From: Bari Zahedi <bzahedi from bccrc.ca>
> Subject: RE: pTopo purification problems
> To: "'Yoram Gerchman'" <gerchman from research.haifa.ac.il>,
>        "methods from oat.bio.indiana.edu" <methods from oat.bio.indiana.edu>
> Message-ID:
>        <B2B905CD6A0E134E9C2347E3B7BF7C8C01D77AA8 from crcmail4.BCCRC.CA>
> Content-Type: text/plain; charset="us-ascii"
>
> This is very little info. It could be your bugs, your lysis, your amp is
> off, it could be many things. Can you provide more info.
>
> To narrow down if the problem is at the plasmid prep level then:
> 1)To start, did you also get blue colonies? Can you get any plasmid out of
> those? This will help determine if there is something wrong with your
> kits/plasmid prep reagents.
> 2)Do you get anything coming out of your preps? Like a smear? If so the
> problem maybe with your lysis.
>
>
> -----Original Message-----
> From: methods-bounces from oat.bio.indiana.edu [mailto:
> methods-bounces from oat.bio.indiana.edu] On Behalf Of Yoram Gerchman
> Sent: Thursday, September 10, 2009 12:55 PM
> To: methods from oat.bio.indiana.edu
> Subject: pTopo purification problems
>
>
> Greetings netters
> I have recently made an attempt to clone a short gene into pTopo (using
> Top10 competent cells). i did get white colonies on LB-Amp-X-gal, and they
> grew nicely on liquid LB-Amp, but after plasmid purification I get no DNA
> (tried already 2 different kits). Any ideas as to how remediate this
> situation?
> Many thanks
> Yoram
>
>
>
> ------------------------------------------------------------------------
> This message was sent using IMP, the Webmail Program of Haifa University
>
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
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>
> ------------------------------
>
> Message: 4
> Date: Fri, 11 Sep 2009 13:09:20 +0100
> From: Duncan Clark <blackhole from abuse.plus.com>
> Subject: Re: pTopo purification problems
> To: methods from net.bio.net
> Message-ID: <M1Be0yAw3jqKFAgO from abuse.plus.com>
> Content-Type: text/plain;charset=us-ascii;format=flowed
>
> Historians believe that in newspost
> <mailman.937.1252617236.21502.methods from net.bio.net> on Thu, 10 Sep 2009,
> Yoram Gerchman <gerchman from research.haifa.ac.il> penned the following
> literary masterpiece:
> >Any ideas as to how remediate this situation?
>
> Screen colonies by colony PCR using M13 forward and Reverse primers.
>
> Toothpick colony into 20ul water, swirl and grid on fresh plate using
> same toothpick. Heat kill 100C for 5mins then use 1ul into a PCR.
> 25-30cycles.
>
> No PCR product, assuming controls work, means no plasmid in those cells.
>
> Duncan
> --
> I love deadlines. I especially like the whooshing noise they make as
> they go flying by.
>
> Duncan Clark
> GeneSys Ltd.
>
>
> ------------------------------
>
> Message: 5
> Date: Fri, 11 Sep 2009 08:33:04 +0100
> From: Cathal Garvey <cathalgarvey from gmail.com>
> Subject: Re: pTopo purification problems
> To: methods from oat.bio.indiana.edu
> Message-ID:
>        <468b1a400909110033g31e40763y66b03648714c47e9 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> I recently had a similar problem, where after plasmid prep/enzyme cleavage,
> I would try to Gel Extract a fragment of the plasmid, only to find I had
> none left at the end.
>
> It turned out to be a bad Qiagen kit, it seems some of the buffers can
> cease
> to work right if they are left to age too long, even if sealed. If the pH
> on
> your buffers is wrong, you can expect to lose the DNA during preparation!
> Which might necessitate either some titration or a new kit.
>
> 2009/9/10 Taliaferro, Dwayne (NIH/NIMH) [F] <taliaferrod from mail.nih.gov>
>
> > Um...try it again?
> >
> > On a constructive note...
> >
> > What size is the gene?
> > Did you -tail it?
> > Did you try a control reaction?
> > Perhaps it was a plasmid prep issue?
> > Are you sure the colony you picked was bacteria vs fungus?
> > Are you sure your AMP is working?
> >
> > On 9/10/09 3:54 PM, "Yoram Gerchman" <gerchman from research.haifa.ac.il>
> > wrote:
> >
> >
> >
> > Greetings netters
> > I have recently made an attempt to clone a short gene into pTopo (using
> > Top10
> > competent cells). i did get white colonies on LB-Amp-X-gal, and they grew
> > nicely on liquid LB-Amp, but after plasmid purification I get no DNA
> (tried
> > already 2 different kits). Any ideas as to how remediate this situation?
> > Many thanks
> > Yoram
> >
> >
> >
> > ------------------------------------------------------------------------
> > This message was sent using IMP, the Webmail Program of Haifa University
> >
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
> >
> >
> > _______________________________________________
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> >
>
>
>
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> End of Methods Digest, Vol 52, Issue 7
> **************************************
>



-- 
Dr V K Gupta
Sr Microbiologist (Molecular Biology)
Insect Molecular Biology Lab
Department of Entomology
Punjab Agricultural University
Ludhiana (Pb)-141004- India
M: 09815963210


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