HELP! re-amplification problem

R. John Lye rjl6n at virginia.edu
Wed Mar 7 12:42:58 EST 2001


bo wrote:

> I designed a pair of degenerate primers directed to a specific gene of a
> green sulfur bacterium. After amplification we could detect several bands in
> the gel that match to our target gene according to its expected size. In
> order to obtain a large amount of target, we planned to re-amplify it using
> the same degenerate primer pair used in the first round.

Why not just clone the gel purified band?  That way you can sequence
it to verify that it is, in fact, the correct sequence and you can grow up
as much as you need.

For whatever reason, re-amplification with the same set of degenerate
primers rarely seems to work well in my hands.  Nested PCR works
much better (for me).

John






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