Re: [ccp4bb] Quikchange cloning: Insert length

- Protein crystallography

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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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

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Subject: Re: Quikchange cloning: Insert length
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-12-01

Subject: Re: Quikchange cloning: Insert length
From: Dima Klenchin klenchin {- at -} FACSTAFF {- dot -} WISC {- dot -} EDU
Date: 2008-12-01

>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.

Here, the maximum we've tried was 3 kbp and it worked just as well as with
the the smaller fragments. Most of the Quickchange cloning we did involved
1500-300 bp and no obvious differences in success rate vs size come to mind.

Dima

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Next message:
Subject: Re: Quikchange cloning: Insert length
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-12-01