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Re: [ccp4bb] Quikchange cloning: Insert length
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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999
Previous message:
Subject: Re: O/T: can a protein which dimerizes in solution crystallize as a monomer?
From: "Fischmann, Thierry" thierry {- dot -} fischmann {- at -} SPCORP {- dot -} COM
Date: 2008-12-01		Next message:
Subject: Re: O/T: can a protein which dimerizes in solution crystallize as a monomer?
From: William Scott wgscott {- at -} CHEMISTRY {- dot -} UCSC {- dot -} EDU
Date: 2008-12-01


Subject: Re: Quikchange cloning: Insert length
From: Michael Giffin mijogiffin {- at -} GMAIL {- dot -} COM
Date: 2008-12-01

Have you seen these papers:

M Geiser, R Cebe, D Drewello, and R Schmitz. Integration of pcr
fragments at any specific site within
cloning vectors without the use of restriction enzymes and dna ligase.
Biotechniques, 31(1):88–90, 2001.

W Wang and B A Malcolm. Two-stage pcr protocol allowing introduction
of multiple mutations,
deletions and insertions using quikchange site-directed mutagenesis.
Biotechniques, 26(4):680–682, 1999.


If i recall correctly, Geiser el al inserted a >1kb fragment with a
modified Quickchange method.


On Mon, Dec 1, 2008 at 12:54 PM, Raji Edayathumangalam
wrote:
> Hi Folks,
>
> Sorry for the non-xtallo posting.
>
> I am curious to hear what is the longest insert anyone has cloned using a
> modification of the Quikchange cloning strategy. Basically,
> ligation-independent cloning by strapping on homologous regions of the
> vector onto the primers which also generate the initial PCR product. I plan
> to proceed with my insert which is ~ 2kb and am curious to get some feedback
> if you have successfully cloned inserts > 1.5kb using the above strategy.
>
> Many thanks.
> Raji
>

Michael Giffin
The Scripps Research Institute
Department of Molecular and Experimental Medicine
10550 North Torrey Pines Road, MEM-131
La Jolla, CA 92037
email: mjgiffin@scripps.edu
lab: 858-784-7758
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