Berglund:Touchdown PCR

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Explanation

Touchdown PCR is useful if you are having trouble with templates (particularly genomic templates). Basically, what happens is that your PCR is not working at the annealing temperature you think it should (maybe your primers aren't quite as specific as you thought). But, if you go too low, you start to get nonspecific annealing of your PCR. What do you do?!? Touchdown allows you to start about 10°C above the annealing temperature and to drop down gradually below the annealing temperature. This allows you to amplify up, most of the time, the template you want. See also PCR additives.

Method

Sample touchdown:

Problem: Annealing temp is 50°C for the primers, but you are getting a double band from your PCR (next time make your annealing temp for your primers more like 55-60°C), product is expected to be about 500bp.

95° 30 sec
95° 30 sec, 60° 30 sec, 72° 1min (3 cycles)
95° 30 sec, 56° 30 sec, 72° 1min (3 cycles)
95° 30 sec, 53° 30 sec, 72° 1min (3 cycles)
95° 30 sec, 50° 30 sec, 72° 1min (25 cycles)
72° 5 min
4° infinity

If this doesn't work, change to:

95° 30 sec
95° 30 sec, 60° 30 sec, 72° 1min (3 cycles)
95° 30 sec, 56° 30 sec, 72° 1min (3 cycles)
95° 30 sec, 53° 30 sec, 72° 1min (3 cycles)
95° 30 sec, 50° 30 sec, 72° 1min (3 cycles)
95° 30 sec, 48° 30 sec, 72° 1min (25 cycles)
72° 5 min
4° infinity