Touchdown PCR protocoll?

[Olivier Gandrillon]

Hi

I have been using the following protocol with some (i.e. not always) 
success to get rid of contaminating bands in my PCR reactions:
95°C for 5 mn, then

72°C for 2 cycles (95°C, 30"; 72°C, 30"; 72°C, 45") then
70°C for 2 cycles
68°C for 2 cycles
66°C for 2 cycles
64°C for 2 cycles
62°C for 3 cycles
60°C for 3 cycles
58°C for 24 cycles

72°C for 10mn
Goto 4°C

Of course the final temperature has to be determined first: you want to 
use a temperature where you know you amplify your fragment of interest 
and the contaminating bands. Hope this helps.

Olivier




Volker Blaschke wrote:
> 
> 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