U.S.E. mutagenesis

[Mark Brandt, Ph.D.]

In article <4v3nfj$9gr at cc-server9.massey.ac.nz>, Jane.Wyatt.1 at massey.ac.nz
wrote:

> Hi guys, 
> I am about to try using the Unique Site Elimination (USE) kit from 
> Pharmacia Biotech, and I am just wondering if there is anyone out there 
> who has used it. I will carrying out the mutagenesis with my cDNA 
> inserted into the pGEX vector ready for E. coli expression.
> Any advice or warnings would be gratefuuly received... I'm sure it won't 
> be as easy as the protocol suggests!
> 
> Thanks heaps,
> 
> Jane.
> 
> E-mail: Jane.Wyatt.1 at uni.massey.ac.nz

I haven't used the Pharmacia kit, but I have used the Clontech Transformer
kit which uses the same method. It generally works very well (I have had
efficiencies as high as 100%, and as low as 10%), and if you follow the
directions, it really is almost as easy as it sounds.

Advice: most of these are obvious, but I will mention them anyway.

Design of the primers is pretty critical. The toggling primers recommended
by the vendor should work, but you will also need a good mutagenesis
primer (my low efficiency reactions occurred in a coding region with a
number of repeated codons and a high GC content).

Don't forget to phosphorylate the primers; the ligation reaction won't
work if you don't (one student forgot, and two others didn't add ATP to
the kinase reaction).

Warnings: I can only think of one off-hand -- I have had a number of cases
in which the final plasmid had significant deletions (in one case 5 of 5
sequences from the same gel; 3 of unknown size, and 2 of exactly 96 bp in
the middle of my coding region). The two I sequenced through the deletion
site had 2 bases of identical sequence on each side of the deletion, so
this may be some form of homologous recombination that occurred in the
mutS strain. I suspect that some apparent negatives (clones that did not
cut with the enzyme I was mutating the unique site to) were actually
plasmids in which the region containing the unique site was deleted.

Hope this helps, and good luck.

Mark Brandt, Ph.D.

-- 
Mark Brandt, Ph.D.
My opinions are my own, but I tend to give them away to anyone who fails to flee fast enough.