I wish to know if this is correct protcol to study RNA from microbial colony lift blots ?
- blotting microbial colony onto nylon membrane
- baking 70c for 5 minutes
- uv cross linking
- incubating with prehybrisation buffer at 60c for 8 hours
- probing for RNA
Yes - the protocol is more or less ok - Points to note:
1) RNA is usually labile - so u can enahnce your capture by using N+ membranes (though that also increases your background.
2) Use strand specific antisense probe as test and sense probe as control to make sure you are detecting only RNA and not DNA (since DNA will also be captured and hybridized). Ratio your control signal from the test for a known gene that is either silenced or expressed (as controls), and compare your result with those ratios.
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