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:[EMAIL PROTECTED] On Behalf Of Mark Collins Sent: Monday, July 21, 2008 9:48 AM To: CCP4BB@JISCMAIL.AC.UK 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 ------------------------------------------------------------------------------ Mark Collins Columbia University Biochemistry & Molecular Biophysics Black Building 259/201 Office/Lab 212 305 1951 (work) [EMAIL PROTECTED]
