In article <7ffjvt$ms0 at net.bio.net>, tjandra at darwin.ucsc.edu (Tjandra) wrote:
> Hi Everybody:
>> I am wondering if there is anybody out there who has a good protocol for
> selecting kanamycin-MX transformants. I am trying to epitope-tag my
> favorite ORF with 3xHA or myc using one of the Longtine vectors, and I
> don't see a high rate of transformants on my geniticin selective plates.
> Is there a 'trick' with transformation protocol using the KanMX system?
>> Many thanks for any info or hints.
>tjandra at darwin.ucsc.edu
Have you looked at some of the original references?
I've been handling strains with kanMX modules using
the protocols in Wach et al. (1994) Yeast, 10:1793-1808
The only "tricks" are;
1) Use G418 (eg. 200 mg/l) in rich (YeP) medium not S.
2) Select transformants either by adding an overlay
of G418 to unselected transformants after 24 h on a plate or
by growing the transformation mix in liquid culture
for a few hours (eg. 4 - 5) in the absence of G418 and
then spreading on a plate containing G418.
I hope that this helps,
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA