Site Directed Mutagenesis within CpG rich promoter - (Sep/07/2006 )

Hi, i am using the site directed mutagenesis kit from stratagene to mutate CpG sites within a CpG island of a gene promoter region.

I am having problems! Both before and after Dpn1 digest i do not see a product on an agarose gel, neither do i get any transformants after XL1 blue transformation.

In total i have had one successful round of site directed mutagenesis but i have not been able to repeat this. Does anyone have any hints when mutating within a CpG rich region. Im not sure if the problem is with my primers not binding. I have tried to change the annealing temperature, elongation time etc but with no sucess. I have also tried to redesign my primers but once again did not have any sucess.

Hope someone can help!

cheers

i'm not expert, but a CpG region needs high denaturation temperature, do u check the activity of the polymerase?

what about methylation at Dpn1 digest site?

Try adding 5% betaine to the PCR mix, which often helps in amplfying high GC regions.

You might also want to run test pcr's using another primer paired with each of the mutating primers. These are less painful to analyze than waiting for transformants, and you might discover which of the primers or which region of the plasmid is giving you trouble.

Hi, i haven't tried betaine - i tried DMSO though and no joy! The pcr test sounds like a good idea - i'll try it,

thanks for your help!