site-directed mutagenesis primer Tm - (Jun/17/2013 )
i'm using partially overlapping primers so the chance for primer dimerization is eliminated. i was wondering if the recommendation of the Tm difference not larger than 5 °C still applies for site-directed mutagenesis primers. or if it even might be better to use primers with about the same Tm. does anyone have some more solid information about this?
Yes, it still applies - you need the primers to have about the same annealing temp so that the reaction for both the forward and reverse is equally efficient. Having said that, the purpose of the SDM as you are probably doing it (i.e. sort of like qickchange), is not really an amplification, but more of a copying reaction, so as long as you get some copies in one direction it should work.
alright, thank you very much!
ok actually i have another question concerning primer design i hope it's ok to post this in this thread.
i'm having trouble finding detailed information about the influence of hairpin formations of primers on the pcr besides the recommendation they just ideally shouldnt form hairpins. this oligo analyzer comes in quite handy for predicting hairpins, but i actually can't estimate to what extent they will be unfavourable for my pcr reaction.
fiddling around with the sequence of the primers sometimes yields a lower hairpin probability, but what if i have a primer that must basically be exactly like the way it is, but has very strong hairpin formation. could i modulate the pcr program somehow? i.e. raising the annealing to the max possible or something?
The most serious hairpins are those which bind tightly to the 3' end, and leave a 5' overhang. These will extend during the PCR reaction, creating oligos which no longer bind your substrate.
It's a shame the tools don't make this distinction. A similar issue for homo and hetero dimers. The other hairpins and dimers are much less of an issue.