"LONG" RT/PCR

[James Gibbs]

Steve Wylie (wylie at CSUVAX1.MURDOCH.EDU.AU) wrote:
: Has anyone tried RT/PCR for RNA of 10KB or longer? I have tried to do 
: this with the potyvirus (BYMV) I work with, but so far no success. 4Kb 
: isn't a problem but anything longer is! I suspect the RT to be the 
: limiting step so have tried M-MLV and AMV RT's, incubating for 3-4 hours 
: but still nothing. Any comments will be gratefully accepted.
: Steve Wylie, Murdoch University, Western Australia 

You can increase the distance that taq polymerase will read by adding a
small portion (1/10 or less, I would guess) of Vent DNA polymerase. This
is what a salesman told me is in most companies Long PCR polymerases (taq
and a bit of vent). The vent is evidently to proofread "stuck"
misincorporated nucleotides.

--
Jim Gibbs                                         gibbs at fas.harvard.edu
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"The truth is where the sculptor's chisel chipped away the lies." -tmbg
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