"LONG" RT/PCR

Duncan Clark Duncan at genesys.demon.co.uk
Tue Nov 22 04:28:13 EST 1994


In article: <3abpup$a2n at dns1.NMSU.Edu>  smori at nmsu.edu (Shahram Mori) writes:
> 
> Steve Wylie (wylie at CSUVAX1.MURDOCH.EDU.AU) wrote:
> : 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


> I would run a gel of your RT reaction( put a film on it) to make sure that 
> You have no RT reaction. It might ( and I suspect it is ) your PCR.
> For denaturing such a large fragment you need at least a minute of
> denaturation.

I am not sure about this length of denaturation. The protocols for LA 
PCR actually insist on short denaturation times of the order of just a 
few seconds for 30-40kb targets, the hypothesis being that heat plus 
the alkaline buffer in the PCR depurinates the DNA rapidly during 
denaturation. This in turn stops the polymerase extending.

Duncan 
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