SAGE

Kathryn Coyne kcoyne at UDel.Edu
Sat Mar 4 11:20:50 EST 2000


Thanks for the input, Gys. To clarify, I am using biotinylated primers.
My problem is not in removing the primers prior to concatenation, the
problem is in generating the 102bp ditags before NlaIII restriction.
I've been trying to troubleshoot and have come up with the following: 
my RNA and cDNA are of good quantity and quality based on gel
electrophoresis; the kinase and ligase reactions seem to be working
properly based on the test I did with the linkers by themselves. The
only parts of the process I haven't tested are the initial NlaIII
reaction, the BsmFI restriction, and the Klenow fill-in reaction.  The
restrictions are easy enough to check out, what about the Klenow? I
admit it's been in the -20 for about a year, but the date is still good.
It is also an exo(minus). (I'm assuming that the excess linkers are not
being "blunted" then, since they have 3' overhangs, and that their
complementary ends will ligate much more efficiently than the blunt
ended ditags?). Maybe I should try T4 DNA polymerase. I also read
somewhere that I could just throw in some Pfu and incubate at 70C for a
few minutes to blunt the ditags instead of using Klenow. Has anyone else
tried this?  Any other ideas?
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