4 replies to this topic

[microgirl]

Hi all, I'm using someone else's PCR protocol - there are two annealing temps, first 48deg for 20 sec then 55deg for 20 sec. Any idea why there are two temps? Also since I'm seeing non-specific amplification (and I'd usually try upping the annealing temp to get rid of it) any suggestions on which annealing temp to up?

[xiaoposhi]

microgirl, on Sep 17 2009, 09:48 AM, said:

Hi all, I'm using someone else's PCR protocol - there are two annealing temps, first 48deg for 20 sec then 55deg for 20 sec. Any idea why there are two temps? Also since I'm seeing non-specific amplification (and I'd usually try upping the annealing temp to get rid of it) any suggestions on which annealing temp to up?

It sounds to me like a touchdown PCR...Is that 48 degree for 20sec, 10 cycles, then 55deg for 25 cycles..something like that.?

[gleb.kudr]

xiaoposhi, on Sep 17 2009, 12:20 PM, said:

20sec, 10 cycles, then 55deg for 25 cycles..something like that.?

In touchdown temperature is decreased in every cycle, not increased.

[microgirl]

It sounds to me like a touchdown PCR...Is that 48 degree for 20sec, 10 cycles, then 55deg for 25 cycles..something like that.?
[/quote]

No - it's not touchdown, the whole 45 cycles have 2 annealing temps!

[LabDiagMol]

Double-stage PCR amplification strategy is widely used specially among those difficult amplicons. But your protocol seems a little bit "ilogical".
I mean, If you start at low annealing temperature then you will favour inespecific products which will be amplified in later cycles (despite a higher annealing temperature). You must make sure that your PCR protocol gives specific products at initial cycles to put them in a fovourable situation.

We sometimes use two annealing temps: 66ºC, 1' (x15 cycles) + 58ºC, 1' (x35 cycles). This is a high performance protocol!
I hope it helps you