Quikchange mutagenesis primer and PCR design - (Nov/21/2007 )
I want to do quickchange site directed mutagenesis. Is it necessary to buy purified primers for this- the kit says you must but my boss says it is not necessary since the primers are only about 30 bases long. Has anyone tried this without primer purification? Also, The instructions for the PCR say that the annealing temperature is 55 C. Is this standard or should I change this based on the melting temperature of my primers. Any help will be appreciated.
We normally order primers with standard desalting and it works for mutagenesis. We follow the protocol from stratagene even for annealing temp.
Sometimes we alter the annealing temp. Try to design your primers according to the quickchange manual and calculate the melting temp online with the tool found on stratagene's website. If you have a temp lower than 78 there and you have no other option (make longer primer or so), you could try and lower the annealing temp in your PCR.
If SDM with annealing @ 55 doesn't work we lower the annealing temp and do a PCR for 40 cycles and see if we get some product, if we do: we proceed with this annealing temp, and if this keeps failing even for extremely low annealing temps (happened once in the past) we redesign the primer (btw: we don't have a gradient cycler, if you have one: try severall temps at once).
I have used standar desalting primers too with no problem.