cloning problems

Daren Rice drice at kumc.edu
Fri Jul 12 10:53:23 EST 2002


I am having trouble subcloning a cDNA our lab is interested in.  I used
PCR to generate the cDNA and then sequenced it to confirm there are no
errors in the sequence.  When I clone it using DH5a bacteria, the
resulting plasmid I get back has mutations in the cDNA sequence. Each
individual clone I pick has different mutations from the others.  The
mutations are mainly point mutations that change a single amino acids,
but sometimes I get single base deletions or insertions that shift the
reading frame, and once I even had a rearrangement.  I have tried other
bacterial strains such as GM33- and M1H, but am still having no luck.
We have just ordered Stratagene's SURE bacterial strain which has some
mutations in genes involved in DNA repair and recombination that should
make them less susceptible to changing my DNA.  If anybody has any
tricks for cloning difficult DNA, I would greatly appreciate them.

Thanks,
Daren Rice
drice at kumc.edu




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