Touchdown PCR: general question?
I am a little confused about the principal of touchdown pcr. If initial PCRs fail, a touchdown PCR is employed in which the initial annealing temperature is higher than the one used in the regular PCR, but lower than Tm? if this is the case then why not conduct a regular PCR at that initial touchdown PCR temp. to avoid any nonspecific binding that occurs due to the subsequent lowering of annealing temperature in touchdown? thanks for the help
- 1 decade agoFavorite Answer
Im pretty surprised that touchdown PCRs are still used. The idea is that you perform another heat shock right, but at higher temps. This could allow for the amplification. The reason it is not done predominately is because some DNA can not take that high of temps. The regular PCR temp is specific to whichever vector you are trying to amplify. Each vector/gene/plasmid/ or whatever you are trying to do a PCR on has a different heat shock point that is ideal for it. This is why I assume that they do regular PCR's. If you are doing PCR's on multiple vectors, then I don't know why you can't do touchdowns, but if you are just continually doing PCR on the same thing, like for research, the PCR temps are always the same because that is the optimal temp for that specific sample.
Hope this helps.
- John RLv 71 decade ago
I still do touchdowns on occasion, when I have a PCR that doesn't work as I expect. The idea is to start at an annealing temp higher than you expect the primers to bind, then drop it by something like 2C every couple of cycles. At some point, you'll get a small amount of very specific binding, which will give you some product of the right kind. Even if you get some non-specific binding later, the thinking is that your first batch of correct product will keep you a couple cycles out in front in the product race. See, you would do a touchdown when for some reason you're not sure where the optimum binding temp is. Nowadays with gradient blocks you can just set up a test run the first time you do a particular primer/template combination, and then you have the information you need.