Hi,
I'm currently working on PCR optimisation for a ~1kb target sequence that contains a large number of inverted repeats. I am using the KAPA HiFi HotStart ReadyMix (https://www.kapabiosystems.com/assets/KAPA_HiFi_HotStart_ReadyMix_TDS.pdf).
SnapGene estimates my primer melting temperatures at ~54 and ~62 degrees respectively. Trouble is, I've also performed a temperature gradient for annealing temperatures from 66 degrees (which is the recommended temp for the KAPA mix) up to 80 degrees (!) without affecting the amplicon quantity as visualised on an agarose gel.
The KAPA mix data sheet does mention that typically annealing temps are higher due to a higher salt concentration, but I am very surprised (and confused) that the PCR is still working when annealing temperature is higher than the extension temperature (!!).
I'm wondering - why is this happening? Has anyone come across this situation in the past?
annealing temperature above extension temperature must always work because if the primers anneal above extension then they will anneal at the extension temperature. Apart from the salt concentration affecting the Tm the Tm is a temperature at which 50% of primer will anneal if possible but primer is in huge excess so even at sub optimal annealing temperatures enough primer will anneal to give a very effective pcr reaction and at later cycles in a normal pcr the amplification is not as efficient as in the early cycles so you may just have a less efficient pcr over more early cycles giving the same amount of product
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