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.

My mind's made up. Don't confuse me with the facts!

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