Query regarding primers for quick change mutagenesis - (Mar/26/2013 )
I have designed the primers for quick change mutagenesis in agilent software....I checked the heterodimer, homodimer and hairpin in one oligo analyzer...http://eu.idtdna.com/analyzer/applications/oligoanalyzer/. In that i found out that
Typically for quikchange you use primers that are completely complementary to each other, so I would expect there to be a lot of heterodimerization. You need to optimise the annealing temperature quite well for this protocol to work.
Another option is to use primers that don't completely overlap...
I don't think -9 kcal/mole is terrible. I usually shoot to keep everything under -9 kcal/mole for all of my primers. bob1 is right that they are completely complementary so they will obviously form dimers. But, you need to remember you are not trying to amplify for purification where you need tons of product. You just need enough to transform into a few bacteria so you get colonies. Thus, I use 50C for annealing ensuring I will get the right product somewhere, even though there will be dimers and non-specific priming.
Agilent even states that if you run your PCR out on a gel, you may not even see a product, but transform anyways, because it should still work. It just doesnt seem right, but I can attest that "no product" reactions can still have the mutation.