Thermo RT's & cDNA library (hi G+C)
graham at biodec.wustl.edu
Fri Apr 26 16:24:07 EST 1996
Having a 65% G+C content in the RNA target of one's cDNA synthesis
reaction suggests the possible benefit of using a thermostable
reverse transcriptase and an elevated extension temperature.
My intital efforts have yielded a reasonable first-strand synthesis
with AMV RT (ie. ~10% of target RNA) at 45C using random hexamers, however
converion to double-stranded material has been dismal at 14C (ie. 25%).
I have seen the suggestion of the use of Tth (Retrotherm RT) polymerase
in combination with MMLV RT in a two step first-strand synthesis intitially
at 37C and then finishing at 70C.
Does anyone have suggestions on how to improve the efficiency of my
sad second-strand synthesis? There should be plenty of random primers
left in the reaction. Apparenlty the DNA polI used for the second
strand extension at 14C has poorer processivity than AMV at 45C on the
RNA template. If I were to throw in a Taq or Vent polymerase, I would
need to cycle down to a low (eg. 14C) and slowwwwwwlly ramp up to a
nice destabilizing 70C or so to move through significant secondary
structure. Any ideas of experience with such a regime?
J. Graham PhD
Washington University of St. Louis
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