PCR trouble!!!

Bjoern Voldborg voldborg at biobase.dk
Thu Feb 8 07:15:51 EST 1996


Hello out there,

I am trying to make a PCR fragment of aprox. 5.5kb, and introducing a restriction site in both ends of it. The 5'end is highly GC rich, and the 3'end very AT rich. The hole thing is made with a cDNA obtained from an RT extension using a cDNA 1st strand synthesis kit from Boehringer Mannheim. using a 15mer dT primer. With this template I am using two primers, both 33 mers where 25 bases are komplementary to the cDNA, and the rest is the restriction site and some closing basepairs. I am using AT pairs for th
e 5' primer and CG pairs for the 3' primer.
I perform the PCR with TaqPlus DNA polymerase from stratagene, using elongations cycles from 6 to 10 min, but everytime all I get is a smear, and not one single distinct band when the PCR product is run on an agarose gel.
Does anybody know what I am doing wrong, any help will be apreciated

HELP!! 
Bjoern
voldborg at biobase.dk
  




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