Following various suggestions posted here or received by email, I tried
the following experiments to see if I could eliminate the ghost band that
I very often see below the correct product.
Briefly, all attempts failed to yeield a solution:
- PCR with only one primer gives no product whatsoever.
- PCR with a 5-fold higher primer concentration (of both primers) gives
about twice as much product, but the proportion of correct and incorrect
products remains the same.
- PCR with a 5-fold lower concentration of primers gives no visible products
(32P overnight with screen).
- The quality of a few particularly "bad primers" was tested by acrylamide
gel electrophoresis of the 32P-kinased primers. They appear to be
essentially pure, probably 99+% (eyeballed).
- Amplification was done with two pairs of primers in the same reaction.
The "good" pair gave one band, the "bad" pair gave both its correct and
Pierre Jelenc pcj1 at columbia.edu
Columbia University, New York