PCR PRIMER DESIGN HELP!!

Wenfu Wang biolc4 at jetson.uh.edu
Tue Sep 27 10:15:11 EST 1994


Hi netters,

Currentlly, we are trying to make a heat-shock construct with which we can
ectopically 
express our interested gene(cDNA) in C elegans. We have two questions: Due
to the lack of helpful restriction sites,  we want to PCR amplify the full
length gene generating a restriction site on the ends (via primers) for
subcloning into the actual expression vector. Question: How long should a
primer be (with one mismatch as to eliminate an upstream ATG) as to assure
proper amplification, and, will 50bps of extra bases from the plasmid
vector (in our 3' UTR downstream of the STOP CODON) impede expression of
our gene????   Thank you for your kind attention.  



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