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 
-- 
    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




More information about the Methods mailing list