LA-PCR

Keith Keith
Wed May 10 10:24:36 EST 1995


mmg6 at psu.edu (mmg6) writes:

>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.


Look at Tessier et. al.: Anal. Biochem. 158:171-178 (1986) and
Grimaldi et.al: Nuc. Acid. Res. 22:2311-2317 (1994) for ligation conditions.
--
Keith Grimaldi, Dept. Oncology
                University College London   TEL: (0)171-636-8333  ext:3419
                91, Riding House Street      FAX: (0)171-436-2956 
                LONDON, W1P 8BT, UK.        k.grimaldi at ucl.ac.uk 



More information about the Methods mailing list