PCR library cloning

flo at UNIXG.UBC.CA flo at UNIXG.UBC.CA
Wed Jul 7 00:37:14 EST 1993


I'm new to the network and I'm looking for some pcr help.  I'm having
trouble cloning a library of cDNAs amplified by pcr.  I have a HindIII
restriction site at either end of my primers and have tried cloning
(HindIII or blunt) into HindIII or SrfI( blunt a la Stratagene) cut
plasmid.  In the past I have cloned lots of individual pcr fragments with
success, however usually only get a few (enough) recombinants.  As I am
trying to make library now, I need better efficiencies.  

I am amplifying with Taq polymerase and get nice S1-insensitive smear of
cDNAs.  The samples are phenol-chloroform extracted, precipitated, then cut
in appropriate buffer.  cDNAs are ligated to vector with NEB ligase + NEB
buffer (which ligate lambda-HindIII fine) then electroporated into
JM101(5X10E8 per microgram pTZ).  Unfortunately I rarely get more than 100
colonies.  I was hoping for several orders of magnitude more !

Any suggestions or good references ?


Thanks a lot,

Roger


Roger Graham

Dept. Microbiology                              email : flo at unixg.ubc.ca
University of British Columbia                          
Vancouver, BC, Canada  V6T 1W5                  FAX : 604-822-6041




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