primer design for site-directed mutagenesis - (Aug/15/2007 )
three yeras back, I had generated a point mutation in a cDNA and had expressed the mutated protein in E.coli.
Now I want to make trangenic construct of the same mutation, can I use the same set of primers and amplify the cDNA using a new template (plasmid expressing the wild type protein plus having promoter, UTL and poly A site ) compatible for transgene expression ??
If you made the construct 3 years back, retrieve it instead of making a new one. And if you need to clone it into another vector, try to do it from the original vector by restriction sites.
i already have the old construct as i had isolated a lot of plasmid DNA, but this wont support transgene expression.
But, this time I need to make the transgenic construct of the same point mutation, please note that I have the wild type [a gene] construct (which has been used to express transgene [a gene] recently) so i can use this transgene construct [a gene] to generate the point mutation.
My question is, can I use the same set of primers which i got synthesiszed 3 years back ? i think we can use it, just want to be sure
It should work, give it a try.