Touchdown PCR protocoll?
vblasch at gwdg.de
Wed Feb 28 05:02:51 EST 1996
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!
Dr. Volker Blaschke
Depts. of Dermatology, Biochemistry
Georg-August-University, Goettingen, Germany
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