varyc.mmcri at mmc.org wrote:
: Hi all,
: I've recently cloned a protein that binds (with differing affinities) a family of structurally related ligands. I have the sequence in pMalC2 (expression) and pGemT (original
: cloning from PCR). I'd like to subject the sequence to random discrete mutagenesis in order to obtain a family of related proteins that possess altered relative
: specificities and affinities for the family of ligands.
: Is there one or mure easily executed mutagenesis techniques that people might recommend? I have a decent screening assay.
: Thanks in advance for any help.
: Cal Vary
Try the Promega's *Altered site II mutagenesis system*.
It makes use of a knocked out ampicilin gene and a functional tetra gene.
Once you have cloned your gene of interest in pALTER, you make it single
stranded. You then anneal the oligo (mutated primer and make sure it's
phosphorylated) to your gene. At the same time you anneal your amp repair
primer to teh vector -> Synthesize the 2nd strand with using T4DNA POl and
then ligate using T4Ligase. The ds vector should have the mutated
nucleotide(s) in it.
C A N A D A
Program in Molecular Biology
Department of Chemistry and Biochemistry Box 3C
New Mexico State University
Las Cruces NM