How to determine PCR annealing temperature
Posted 27 October 2004 - 11:05 AM
Posted 27 October 2004 - 12:32 PM
Hi, The simplest way is:
Hi, somebody knows how to estmate T annealing of a set of primers?
Tm = 2(A + T) + 4(C + G) degrees. It is roughly right. But lot of softwares calculate Tm for you.
Posted 28 October 2004 - 11:47 AM
Posted 28 October 2004 - 11:28 PM
Usually for standardising a pcr annealing temperature we start from a temp. 5 degrees lower than the Tm.
but the one that I'm doing now has an annealing temperature of 10 degrees higher than the Tm ( since at lower annealing temp. I got many nonspecific products together with the desired one.)
Posted 29 October 2004 - 03:00 AM
Posted 04 November 2004 - 04:27 PM
one has 340 bp Tm>100
one has 20 bp Tm=65.9
The PCR product shoule be around 850 bp.
What annealing temperature should I use?
What other suggestion for me, such as the time for extension?