Anyone successfully doing RNAse protection Assays?
dotzlaw at cc.umanitoba.ca
Fri Mar 1 11:29:31 EST 1996
In article <3135F21D.DA0 at unixg.ubc.ca>, genes at unixg.ubc.ca wrote:
> I have started doing RNAse protection assays using an Ambion kit(RNAse
> II). I can't seem to get it to work. The probe is 600 bp and is made
> from a pGEM plasmid containing an insert. The target is made from a
> longer sequence that is in another plasmid (probe was actually
> subcloned from this longer sequnce). I am trying to titre the reaction
> so as not to use up my total RNA sample that is limited. The control
> for the kit works fine. Can anyone give me some pointers on making the
> probe or the target sample and getting this to work.
> Any help will be greatly appreciated.
I've done a few. ok - thousands.
First thing that comes to mind is are you absolutely certain that your
target is sense and the probe antisense?
What do you mean exactly that it "doesn't work." No protected fragment of
the right size but smaller bits, excess probe doesn't digest away, no
signal at all?
Does your probe look ok on a gel?
A couple of tips:
600 bp is a largish probe in RNAP. 200 - 300 is ideal. See if you can
cut into your insert to generate the riboprobe.
Make certain that you linearize the probe template with an enzyme leaving
a 5' extension. The polymerase can loop back on 3' and (less effectively
but it happens) on blunt ends, resulting in a protected probe. Make sure
that the probe template is completely linearized. Play with hyp temp - 42
C is a good place to start.
Let me know if you have any further questions.
University of Manitoba, Dept. Biochem. Mol. Bio.
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