I need to do a site-directed mutagenesis of a DNA insert in a pCAX plasmid with a CAG promoter.
I have used "QuickChange II XL Site-Directed Mutagenesis Kit" (Agilent Technologies) to make a point mutation of DNA in a mammalian plasmid, but I have never obtained the desired result.
I have tried to change parameters (I have increased time of initial denaturation) and volume of buffers (I have used the highest volume of DMSO suggested), as suggested by the kit, but no desired result obtained.
Can anyone give me advices?
Thank you.
I doubt that it has anything to do with the source of the target DNA, in other words it doesn't matter that you are working with CAG promoter or anything else. However there may be a size effect such that efficiency goes down as the zie of your plasmid increases. Quickchange can sometimes be tricky. I would be sure that you can get all the controls to work well first. Also double check that you designed all your primers correctly.
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