Methods Digest, Vol 38, Issue 2-pCambia 1201

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Sat Jul 5 07:20:54 EST 2008


It is really depressing when you donot get the desired results. If the
plasmid is good sometime you loose sites that are involved in
restriction followed by ligation, others remain unaffected. It also
happens if the plasmid is isolated using phenol chloroform extraction
using old preparation of phenol ( gets oxidized with time and makes
restriction sites resistant to cut). I suggest go back to your
original plasmid which you acquired from any other source, transform
into DH5-alpha and isolate plasmid using alkaline lysis method (
solution P2 should not be old- absorbs CO2 from atmosphere to result
decrease in pH). This will also enhance plasmid yield. (Do not use
plasmid purification kit based upon glass memebrane or glass powder.)
Try cutting this plasmid.I am sure if recieved right plasmid, you will
be successful in restricting all sites. It must work now and if does
not just forget and  order for new preparation from CAMBIA- (available
freely as DNA spot on filter paper- Cut the disk, elute DNa with TE
use eluent for transforming DH5-Alpha). Tell me about results.
Alternately move to pCambia 130. All the best.

On 7/3/08, methods-request from oat.bio.indiana.edu
<methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
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>   1. pCambia1201 question (James J. Campanella)
>   2. Re: protocol for membrane receptor isolation
>      (Dr Engelbert Buxbaum)
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> ----------------------------------------------------------------------
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> Message: 1
> Date: Tue, 01 Jul 2008 19:34:50 -0400
> From: "James J. Campanella" <campanellj from mail.montclair.edu>
> Subject: pCambia1201 question
> To: methods from magpie.bio.indiana.edu
> Message-ID: <fc7ffef0256c.486a86da from montclair.edu>
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>
> Dear fellow workers in the mines of science,
>
> I have been tryingto work with the pCambia1201 plasmid for the lastseveral weeks, but ithas been driving my lab a bit crazy. My problemis that it does not wantto digest with most endonucleases we've tried.I simply can not use theplasmid if I can't cut it, so I have aproblem. It was suggested to meto try transferring the plasmid to DH5cells, which I did to no avail--the resultant plasmid still does notcut with almost every enzyme in mylibrary. I was finally able to getit to cut with XhoI and PvuII, buteven those were only partialdigests.
>
> Does anybody have anyadvice on working with this thing? The literaturedoes not suggest thatthere is even a problem, and the group thatengineered it could onlysuggest "trying all your enzymes until youfind one that cuts", which isa bit impractical.
>
> Thanks for any help,
>
> Jim Campanella
>
> -------------------
>
> James J. Campanella,
> Associate Professor,
> Department of Biology and Molecular Biology
> Montclair State University
> 1 Normal Avenue
> Montclair, NJ 07043
>
> Alternate email address: jcamp from alumni.uchicago.edu
>
> Ph: 973-655-4097
> Fax: 973-655-7047
>
>
>
> ------------------------------
>
> Message: 2
> Date: Thu, 03 Jul 2008 10:14:53 -0400
> From: "Dr Engelbert Buxbaum" <engelbert_buxbaum from hotmail.com>
> Subject: Re: protocol for membrane receptor isolation
> To: methods from net.bio.net
> Message-ID: <op.udpy64a366vu6s from bengelbert-dm.rusm.rossu.loc>
> Content-Type: text/plain; format=flowed; delsp=yes;
>        charset=iso-8859-15
>
> Am 25.06.2008, 06:18 Uhr, schrieb WS <novalidaddress from nurfuerspam.de>:
>
> > Just wonder if membrane proteins get solubilized without detegent?
>
> No. Membrane attached proteins can be washed of membranes by high salt
> (100 mM Na2CO3 pH 11.5 or 250 mM KI or 2 M NaBr), but transmembrane
> proteins require detergents.
>
> > Will eg 0.5% TX100 disrupt the antibody-recptor complex?
>
> That depends on the nature and concentration of the detergent and on the
> antibody-antigen interactions. Note however that in immunoprecipitation
> studies even relatively harsh detergents like SDS are frequently used to
> ensure specificity. For example, in their booklet on Pansorbin Calbiochem
> recommends the following "superwash buffer": 100 mM NaCl, 50 mM Tris-HCl
> pH 7.4, 2 mM EDTA, 2% Triton X-100, 0.5% SDS. Of course, not all
> antibodies can be used with these conditions, but some titration should
> give you detergent concentrations where specific binding survives with
> minimal background.
>
>
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> End of Methods Digest, Vol 38, Issue 2
> **************************************
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-- 
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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