SuperScript II cDNA prep ... can I merge steps?
Ian A. York
iayork at panix.com
Wed Jun 19 12:14:28 EST 2002
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?
I don't really understand the point of that magical two minutes, but I'm
also reluctant to waste my time and RNA comparing right now, so I'd like
to see if someone else has tried it.
Ian York (iayork at panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
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