Touchdown PCR protocoll?

[Volker Blaschke]

Dear netters,

I am looking for advice on how to do touchdown PCR. My current protocoll 
is 30 s @ 95 C, 1 min @ 63 C and 2 min @ 70 C.
I should get a product of 1550 bp, which is unfortunately only faint. The 
main product is 500 bp with a huge band. Now, I was thinking that 
touchdown PCR might increase specificity and rid me of the 500 bp 
product. I am not sure if further increasing annealing temp would make 
that much of a difference.

What would you do for touchdown?

I was thinking of starting at 70 C, but I am not sure what the decrease 
in annealing temp should be per cycle ? Should I go down 1 C / cycle for 
seven cycles and continue for another 28 cycles at 63 C, or should I 
decrease at say 0.5 C for 14 cycles?

Any input is most welcome!

   Volker


-- 
Dr. Volker Blaschke
Depts. of Dermatology, Biochemistry
Georg-August-University, Goettingen, Germany