nonspecific primer extension products

Barr Lab Barrlab at MAIL.MED.UPENN.EDU
Wed Feb 19 09:27:11 EST 1997


After following the "RedBook" protocol for primer extension analysis, I
observed bands that were both in the experimental and negative control
lanes. These bands are intense (far too intense to be products from the
transcript of interest), of specific sizes which are not dependent upon
the sequence of the primer ( I get different sized bands for different
primers). Examination of the primer sequences did not indicate that
self-annealing could be the problem. These bands are not observed in the
DNA sequenceing ladders that are generated with the same primers. What
are these things and how are they generated? I am using AMV RTase and
have observed them in total and poly(A+) RNA samples. I am afraid that
they are interfereing with the assay by raising the background within
the lanes and perhaps reducing the efficiency of the RT step. 

	Rich Davis



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