RT PCR woes...

John Brennand john.brennand at gbapr.zeneca.com
Tue Dec 17 05:02:15 EST 1996


ed

one possibility is that some RNA's seem to have "pause" sites for RTase
- possibly due to strong secondary structure in the RNA such that the
cDNA may not get past that point.

so we heat the rna in water at 70oC for 5', quick cool and initiate cDNA
synthesis quickly.  also some RTases are better than others in getting
through these regions - since we started using Life Tech's Superscript
II (no affiliation) we always get good full length cDNA.  we also do an
oligo-dT AND a random Hex primed cDNA synthesis and then pool the
reactions.

hope this may help

john



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