Using a protocol for cDNA cloning from Clontech, I have very little
yield in 2nd strand cDNA product compared to my old protocol.
The clontech protocol uses a mixture of DNA Pol. I, E.coli ligase and
RNase H in a E. coli ligase buffer. Reaction at 16 deg for 2 hours
followed by 1/2 hour at 16 deg with additional 9 units of T4 pol for
My question: couldn't it be better if the reaction performes at 16 deg
for 1 h followed by 1 h at room temp?
My old protocol had this sheme, but it didn't contain the ligase and had
a different buffer as well.
Burkhard Hassel, Heidelberg