"LONG" RT/PCR

[Steve Wylie]

Has anyone tried RT/PCR from RNA of 10Kb or longer? I have been trying to 
do this with the potyvirus (BYMV) I work with but with no success. 4Kb is 
no problem providing the RT reaction is allowed to proceed for 3-4 hr. I 
suspect the RT to be the limiting step so have tried M-MLV and AMV RT's 
with no luck. I don't use a special long PCR kit, I've just added some 
Pfu to the Taq and 8% glycerol and 1.5% DMSO to the mix, as well as 
reducing to denaturation time to 10sec. Any comments gratefully accepted. 
Steve Wylie, Murdoch University, Western Australia