nonspecific primer extension products

martin LEACH leach at bu.edu
Thu Feb 20 17:22:06 EST 1997


looks like my response disappeared somehow..

1. perform primer extension with two primers (30 bp apart) in parallel tubes.
2. perform the primer extension with a sample (e.g. I wanted brain
transcripts) and a negative control (i used HELA RNA)

...specific products will be detected by both primers......exlude everything
that does not have a 30bp larger counterpart...

furthermore...when comparing tissues exclude everything that has a counterpart
in the negative control...

hope this helps

M

Barr Lab (Barrlab at MAIL.MED.UPENN.EDU) wrote:
: 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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