RPA's

Miss. V.M. Hodges vhodges at hgmp.mrc.ac.uk
Fri Jul 5 07:31:45 EST 1996


I've been doing RPA's for a while and I have some recurring problems ( 
although initially everything worked really well):

1. I'm getting very low yields of full length transcribed probe.
I've tried using both PCR product ( with an SP6 primer) and linearised 
plasmid. In addition I've tried carrying out the transcription reaction
at 15 degrees C as well as the normal 37 but with no improvement in probe 
yield.
I'm using the maxiscript kit from Ambion and gel purifying my probe.

2. when I add GAPDH control probe in at the same time as my test probe the 
signal for the test probe is much lower compared to reactions with test 
probe alone.

I've tried several batches of the kit so I know it's not due to enzyme 
problems.
Also has anyone noticed differences in batches of UTP?

If anyone has any suggestions I'd love to hear them.

Thanks
Vivien.



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