northerns

Soon Paik paiks at ari.net
Mon Aug 7 23:01:52 EST 1995


Nigel Kenward wrote
>Soon Paik <paiks at ari.net> wrote:
>>Aardraa Potnis wrote,
>>How do you get rid of background bands of 18s, 28s RNA on the >northerns??
>>I have used all kinds of stringent conditions and still have them !
>>Please help!
>
>How about treating the blot with RNase during the wash step?
>
>Soon Paik
>Lombardi Cancer Center
>


>DON'T !


>Unless you're sure your probe is a 100% match with your required RNA >sequence ! This is an idea used in RNAse protection assays and
>works well only under the conditions required for these assays. If >you're not set up to do RNAse protection assays, keep RNAse well
>out of the way of your Northerns !

Well, I am sorry if I have misinformed you about this. I myself do not have experience with this method. However, I remember many investigators in this center using this method before we all moved to RNase protection assay. In theory, even if there is not 100% match with the probe, RNase A will only nick and degrade the non-matching part and matching hybrids should remain on the blot. Correct me if I am wrong.
Another way of achieving clean blot is to hybridize with a riboprobe at high temperaturem, since RNA-RNA hybrids should be more stable.

Sincerly,
Soon Paik
Lombardi Cancer Center






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