LA-PCR - help!!!!

mmg6 mmg6 at psu.edu
Tue May 9 20:23:56 EST 1995


I have been unable to clone the ends of viral dsRNAs by
ligation-amplification PCR.  I think the problem lies in the ligation
reaction ie. the addition of a 5'P-18nt-NH2 3' primer to the ends of
the dsRNAs.  I am using RNA ligase from NEB and Tth polymerase as both
reverse transcriptase and DNA polymerase. Standard RT-PCR works fine
for the amplification of internal sequences.  Any suggestions would be
greatly appreciated.

Cheers,

Michael



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