LA-PCR

mmg6 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.

Cheers,

Michael

P.S. - the problem is not with the RT-PCR - internal sequences are
easily amplified using Tth-mediated RT-PCR.



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