| | Re: Touchdown PCR question - why does this work?
Saw this and the coincidence is that its running behind me right now. The reason one would run a touchdown PCR, besides the technique sounding as nearly cool as the Sonic Hedgehog gene, is that if you're having trouble figuring out the correct annealing temperature of your primer, this is a way to use primer competition to find the right annealing temperature.
The reason it starts out high is because it tries to find the annealing temperature of your primers and target first before it finds background. By starting high, once it gets to the actual unknown annealing temperature, it will then start producing target product . Using the principle of competitive resources, by the time it gets to the lower unspecific product, the target, which is now in high concentration, will outcompete other product and will be the dominant product.
In regard to primer that hasn't worked before, it could be you were using the wrong annealing temperature in the past.
The con ? Well, its not nearly as clean as if you knew your specific annealing temperature, but having something from nothing is a heck of a lot better !