U.S.E. mutagenesis

Mark Brandt, Ph.D. mark at indy-lv.biomol.uci.edu
Thu Aug 22 21:39:10 EST 1996

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

> 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.

More information about the Methods mailing list