"LONG" RT/PCR

[Shahram Mori]

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
In Focus there is a good article on reagents that inhibit the M-MLV (
Superscript RTase).
The following has been shown to inhibit the enzyme:
1) 0.5 mM spermidine HCL
2) 4mM NaPyrophospahe
3) 2.5mM EDTA
4)17% (V/V) DMSO and 5% ( V/V) formamide
5) 0.005% SDS
6) 70mM GITC
7)160mM Guandine-HCL

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.
Good Luck


 --
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003