Methods Digest, Vol 38, Issue 2

[Virash Gupta]

One thing more- retransform. Pick up 5-6 single clones (Blue clnes on
X-Gal/IPTG) culture individually and isolate plasmid from each
culture. Compare restriction and select the best one.
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)
>
>
> ----------------------------------------------------------------------
>
> 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
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> Ph: 973-655-4097
> Fax: 973-655-7047
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>
>
> ------------------------------
>
> 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;
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> 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