PCR trouble with Arabidopsis gene

Bert Popping Bert.Popping at durham.ac.uk
Sat Mar 11 09:59:50 EST 1995


Dear All,

I tried to PCR genes from an Arabidopsis cDNA library (phage lib.).
It worked fine for one gene but the other causes a lot of trouble:

for this PCR product, *ONE* primer was enough to make it.
Both primers (27mers: 19 exact matches, 1 EcoRI site + 2 additional 
bases at the end) were designed for the same annealing temperature 
(55C).But the forward primer alone was sufficient to produce this PCR 
product(which was not the gene we were looking for). I changed 
parameters like annealing temperature, MgCl2-conc etc. but it did 
not work.Even a 'pre-run' with the delayed addition of the forward
primer did not give a better result...

Any clever ideas ... ?

Bert Popping

Bert.Popping at durham.ac.uk



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