2 replies to this topic

[labgrl]

I was told I need to do gradient PCR on my primers to optimize the temperature. I understand the theory behind this, but don't know how it is actually done? A 96 well plate? Any other hints would be appreciated, too! Thanks!

[vetticus3]

it really depends on the make/model of gradient PCR machine that you're going to use.
check out the manual for your machine (online?).  it'll go into the details on how to set the machine up.
what happens usually is the machine is able to set several different temperatures over the run.  so if you're running a PCR reaction, with an annealing temp of say 55'C.... the gradient machine will run each row at a different temp.  so if you have a gradient of 5'C, the machine will run a PCR reaction with the annealing temp at 50'C, 51'C... to 60'C.   Each different temp should be assigned to a different  row or lane on the machine.  ie if the 0 are where you normally put the tubes for the reaction, and each lane is where the temp changes...

OOOOOOOOOO  50'C
OOOOOOOOOO  51'C
OOOOOOOOOO  52'C
OOOOOOOOOO  53'C
OOOOOOOOOO  54'C
OOOOOOOOOO  55'C

You would place one tube in each lane... let the reaction run,... do a gel noting the temperatre difference, and viola, results.

does this help?

[labgrl]

Yes this does!! Thanks so much for your help vetticus3