Anyone successfully doing RNAse protection Assays?
cashug at aol.com
Tue Mar 12 00:24:13 EST 1996
Just read your note on RNAse protection assays--I've used the Ambion kit
quite a few times with limited success until I changed the way I prepared
the probe. Instead of linearizing my plasmid and purifying it, I followed
Ambion's suggestion of preparing it by PCR. I made an oligo (from 5' to
3'): the T7 pol seq then a 15bp spacer seq then the antisense 20-mer. I
used a regular 20-mer sense oligo and amplified by PCR the area I wanted,
using a plasmid miniprep as template--I used 10ng. Then I used 5ul of the
PCR rxn in the MAXIscript (T7) protocol. Never a problem. This whole
process is listed in the back of 1994 or 1995's Ambion catalog--not the
most recent one. The only change I did was elongate the spacer region to
15bp--the 5bp Ambion had is too small to see as a convincing difference
between undigested probe and protected fragment.
That's been my biggest problem with RPA's--hope this helps!
Let me know how you're doing--good luck
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