2nd Strand cDNA synthesis with Sequenase

[SIT at PARGVA.AGR.CA]

We have successfully used Sequenase for second strand synthesis to produce a
full-length infectious clone of papaya mosaic potexvirus (ca. 6.7 kb) after
first strand synthesis with Superscript (J.Gen.Virol.74,1133-1140 1993). I'm
sure the only limiting factor was the quality of the RNA template which must
be very clean and homogeneous in size (we sucrose fractionated the viral RNA)
since AMV RT has been rated to go upwards of 10kb for first strand synthesis
(claims the supplier). I'm not sure how well Superscript II works for very
long first strands though because it wasn't available when I was making the
clones!

Tim Sit
Agriculture and Agri-Food Canada
Pacific Agriculture Research Centre
6660 N.W. Marine Dr.
Vancouver, B.C. V6T 1X2
CANADA, eh?!