mmg6 at psu.edu
Tue May 9 20:44:46 EST 1995
I am getting really good at separating DNA standards by gel
electrophoresis. However, what I really want to do is amplify the ends
of viral dsRNAs by ligation-amplification PCR. I think the problem
lies in the addition of a 5' P-18nt-NH2 3' oligo to the ends of the
dsRNA using T4 RNA ligase (from NEB). I am using an oligo
complimentary to the above mentioned oligo and internal primers for the
subsequent RT-PCR step for which I employ Tth polymerase as both
reverse transcriptase and DNA polymerase. I would be grateful for any
suggestions concerning conditions for primer ligation.
P.S. - the problem is not with the RT-PCR - internal sequences are
easily amplified using Tth-mediated RT-PCR.
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