Designing primers to clone into expression vector.

[CathA]

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. I have no idea what I'm doing and I'd really appreciate some advice.

Thanks very much!

[CathA]

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?

[mmorgan]

You can either get a kit (I have used Stratagene's QuickChangeII kit and it is quite reliable and fast), or you can do a 4 primer cloning. I'll try to represent it here on the page but I dont know how well it will display, so apologies if it looks like a bunch of mess.

Reaction #1
Forward primer Mutation Reverse
>>>>>>>>> <<<<<MMM<<<<


Reaction #2
Mutation Forward Reverse
>>>>>MMM>>>>> <<<<<<<


You run 2 separate reactions then mix the products and do PCR with the "outside" primers.