final extension real time PCR - (Jul/01/2008 )
Out of lab-lore we do a final extension on our Real time PCR protocol. We do carryout a dissociation curve, but was wondering if it is necessary to carryout the final extension prior? Our amplicon is ~100bp. Does anyone else do this final extension when amplifying such a small sequence? I will probably do the experiment, but wanted some opinions so I can be biased in my expectations.
Hm, personally I do not find it necessary.
I agree with Pallas. Not necessary
w/ 7min 72°C final extension
w/o 7min 72°C final extension
I see no discernable difference between my samples in either run
Red = standards
Black = positive control
Green = negative DNA control
Blue= no template control
Hope this helps, it did for me!
Cheers Randy, I appreciate your input.
I mean, the purpose of the final extension is to increase the sensitivity of classical PCR. Sensitivity of qPCR is high enough to make this step irrelevant.