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Designing primers to clone into expression vector

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Designing primers to clone into expression vector

Postby Jemi » Wed Feb 15, 2012 8:11 pm

Hi,

I'm just doing a piece of work at the moment and I'm a little stuck. I've got a DNA ligase sequence and I've created primers for it to mutate two residues but now I have to design two outer primers to clone the gene into a pET expression vector and I have no idea how!

I've pretty much been staring at a blank page all day but I think I might have just had a breakthrough thanks to trusty wikipedia, on the site directed mutagenesis wiki page it says "Variations employ three or four oligionucleotides, two of which may be non-mutagenic oligonucleotides that cover two convenient restriction sites and generate a fragment that can be digested and ligated into a plasmid, while the mutagenic oligonucleotide may be complementary to a location within that fragment well away from any convenient restriction site.

Am I right in saying that what I should be doing is creating another pair of primers, one upstream and one downstream of my current primers, which cover a restriction site which can be cut to create a sticky end which I can use to insert my protein into a pet vector?

Thanks!
Jemi
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Postby canalon » Thu Feb 16, 2012 3:08 am

Hmmm...
I would say create 2 primers that cover the end of your sequence of iterest and add a restriction site of your choice as an overhang at each end.
Patrick

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