touchdown pcr - (May/10/2009 )

hey can any one plz tell me the details about touch down pcr ........
how it is set up ????????
wat range of annealing can be there?????????
for wat set of primers can this pcr be done ????????? i.e. the permissible limit of tm difference bw two primers????
and wat steps to be included???????
extension step is there or not wen the annealing is changing after every cycle???
plz reply ........

thanks in advance

Touchdown is the same as ordinary PCR, just with a lowering of the annealing temperature at each cycle step. You set it up as you would for a normal PCR, however in the cycling conditions you need to lower the annealing temperature by a small amount (usually about a degree C) per cycle to a pre-determined low temperature. Some machines will do this automatically for you. You shouldn't need to go above 72 deg C (can you tell me why?) for the upper limit of annealing, lower limit could be as low as you like really, but most people leave it reasonably specific.

You can do this for any set of primers. How big a difference do you have in your primers. Ideally they would have very similar annealing temperatures.
You do need the extension step.

bob1 on May 10 2009, 05:46 PM said:

hey thanks ........bt m still left with the same doubts that i had earlier........
and regarding 72 ..... this or above polymerase is well suited to act ......... is it ??

i was confused about to set the range of temp variation ........as TD pcr is to minimise the non specific amplification ..so if one keep a wide varying range than hw cm specificity would be thr..........

plz guide if u wish

thanks

minty on May 11 2009, 03:22 PM said:

Before I go any further, there isn't a character limit, so you can use the whole word... even poorly-spelled words are prefferred to SMS-talk!

Next, have you actually googled "touchdown PCR"? Try it; I just did and came back with over 21000 specific hits. The first page of 10 should all give you the answer to your homework question...

swanny on May 10 2009, 11:28 PM said:

thanks a lot google master.............

knowledge brings confidence but here seems over ... turning in ego

minty on May 11 2009, 06:52 PM said:

Sorry minty, but we have so many homework questions come up, and it does no-one any good if the established members just give out the answers. We're always happy to extend knowledge, but we can't in all good conscience start and extend it, so if you can give us what you've already found out, we'll happily extend, edit, amend, emend and expand!

swanny on May 11 2009, 06:25 PM said:

thanks for your kind generosity..
i had already read a lot about TD pcr ...and than i did it with my set of primers
bt i didnt got any result ....
so i asked this way that may be i have understood smething wrong on my own ...bt..............!!!!!!!!!

i had done TD pcr with a set of primers with tm 61.9 and 67.2...with expected amplicon as 1.2 kb
machine we have in our lab automatically decreases annealing by 0.5 wt every cycle
i set up at 95(3 min),than 95(40 sec), 65(30 sec), 72 (1 min) for 20 cycles wt annealing incrementally decreases by 0.5 every cycle and than 95(40 sec), 65(30 sec), 72(1 min) for 10 cycles and final at 72(8 min).

similarly for 2nd set of primers with tm 59.4 and 66.4 wt expected amplicon as 1.7 kb i set up wt similar conditions...

but unfortunately i didnt got any thing...