Primer design invert pcr - (May/16/2006 )
I want to delete a region from a plasmid (for later on doing homologuous recombination in mammalian cells with this plasmid that will then be linearised with a restriction enzyme I have to introduce and PCR product).
As there are no restriction unique restriction enzymes flanking the region I want to delete, I will try to delete the region by "invert/inverse PCR", meaning that I will amplify the entire plasmid except for my region of interest (my primers are pointing outwards from that region if you know what I mean), afterwards I will digest with DpnI (as you would with site directed mutagenesis), then ligate the product with itself and clone into e. coli.
Has anyone done this? My main questions are if you need to have special designed primers (besides the obvious concers in primer design) and is it neccessary to gel purify after DpnI digestion?
Yes, this can work. You'll need to add a restriction site (plus some 5' extra bases) which does not occur in your vector to the end of your primers, then cut with that enzyme and religate. I'd follow the idea of the Quikchange kits -- use lots of template, a small number of cycles (12-16) and cut with DpnI. You can do that at the same time you are cutting with your selected enzyme, most likely.
I was actually planning on just doing PCR with a proofreading enzyme, and then cutting with DpnI after wich I would go for blunt ligation... this would make the extra cutting unnecessary, and I could just add a restriction site on one primer...
No special requirements on the primer site?
Thanx for your response!
You'll need to phosphorylate your primer if you want to blunt ligate. Also, you'll need to make sure the ends are really blunt -- Taq adds an A often at the 3' end. An enzyme like PFU or Phusion will produce blunt ends.
I have PfuUltra available from previous experiments (I need the smallest possible amounts of mutations), but hadn't considered phosphoylation, thanx for the hint!
I would use the four primer method with heterologous dimerization to create overlapping sticky ends.
Digest away the template with Dpn I and you are done.