Smeary touchdown PCR gel.

[Dietmar]

Dear Stephanie,
the idea of touch down PCR is to combine high efficiency with 
high specificity. For this purpose, PCR is started at high 
annealing temperatures (about 15-20C above Tm). So, my first 
suggestion would be to start your PCR at a higher annealing temp. 
(about 70-75C). A 2-step-PCR is good enough. After 20 cycles one 
may end up at the optimal annealing temp. (about 5C below Tm, 
i.e. 55C).
Did I get it right? Do you really supply 10 mM dNTP mix in your 
PCR reaction? If that is true you have to decrease the conc.! 
Normally,the final conc. should be 20 - 200 mircomolar!
You may use a 2-step-PCR if the Tm of the primers are high,i.e. 
near the extension temp. (72C). At these high annealing temp., 
Taq is already synthesizing DNA with high efficiency, and an 
additional extension step is unnecessary.
If low annealing temps are used (below about 65C), this extra 
extension is needed, especially if your PCR products are long 
(i.e., more than a couple of 100 bp).
Hope this helps.

Dietmar Kahle, Ph.D.
Eppendorf-Netheler-Hinz GmbH, Hamburg