I'm working with a 500bp pcr insert cloned into the TA cloning kit vector.
I'm having terrible problems trying to get any probe off of it. I'm using
the sp6 polymerase to transcribe antisense off of a CsCl prep, linearized
with Eco RV. The Genius kit recommends 1ug of DNA to start with, and you
should recover 10ug of probe. Ha! When I do a simple dot blot on my probes
I get a 10-100fold less concentration than the kits control labeled RNA.
When I run a tenth of the reaction out on a gel, I don't see a thing. The
first time I used the kit with an insert in poly Blue Script I got a
beautiful probe. Great band on a gel and close to the same concentration as
the control labeled RNA.
So I'm wondering, is it the vector, the pcr insert, polymerase failure (new
kit). What if I used twice as much DNA to start out with? Please help if
you can. Thanks