Another "new school cloning" reference:
Klock, H.E., Koesema, E.J., Knuth, M.W. & Lesley, S.A. "Combining the polymerase
incomplete primer extension method for cloning and mutagenesis with microscreening
to accelerate structural genomics efforts." Proteins (2008) 71, 982-994.
published online 14 November 2007 (doi:10.1002/prot.21786).
http://dx.doi.org/10.1002/prot.21786
Regards,
Mitch
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Mark Collins
Sent: Monday, July 21, 2008 9:48 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] Expression vector with NdeI-ClaI sites
Or do the new school cloning, SLIC (Sequence & Ligation Independent
Cloning) and "subclone" into any position in a vector.
This method uses a PCR product and vector, the PCR primers have 20-40bp
overlap with a region in the vector. Mix cut and purified vector with PCR
product. Digest with T4 polymerase, quench, and transform.
When I do the PCR in the morning I have clonies the next day.
REF:
Harnessing homologous recombination in vitro to generate recombinant DNA
via SLIC
Mamie Z Li & Stephen J Elledge
Nat Meth V4(3) pp 251
Mark
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Mark Collins
Columbia University
Biochemistry & Molecular Biophysics
Black Building 259/201 Office/Lab
212 305 1951 (work)
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