SuperScript II cDNA prep ... can I merge steps?
andrew.bettany at bbsrc.ac.uk
Thu Jun 20 02:36:25 EST 2002
Maybe I have misread my SuperScript protocol but I use a mastermix routinely
and get cDNA (of course, I might get more if I am doing it wrong . . . .).
I denature the RNA and random primers for 10 minutes at 65oC and then add to
an aliquot of mastermix containing all the necessary components (including
the SuperScript). I then incubate this mix at 25oC for 10 mins and 42oC for
60 mins. As I said, it works for me - and is considerably less painful than
the method you describe!
"Ian A. York" <iayork at panix.com> wrote in message
news:aeqe5j$171$1 at reader1.panix.com...
> SuperScript II and other RTs have as part of their protocol to assemble
> all ingredients but the RT, incubate for a couple minutes at 42 (or 37)
> oC, and then add the RT.
> That's a pain, when you have a bunch of samples. It would be vastly
> easier to include the RT in the master mix at the beginning (or rather at
> the second step, because you'd still need to do the denaturing and
> snap-cool step). Has anyone tried adding the RT at that point rather than
> waiting for 2 minutes and adding it?
> A further complication is that I'm using random primers for the cDNA,
> which calls for a 25 oC 10 minute incubation before shifting to 42 oC for
> 2 minutes, then adding SuperScript ... would that 10 minutes at 25 oC
> damage the SuperScript?
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