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?
You can use standard lithium acetate protocols (e.g. Geitz, D., et al. (1992)
Nucleic Acids Res. 20, 1425) for transformation with kanMX; the only
difference is that you need to grow the cells non-selectively on plain YPD
(without drug) before plating on YPD + G418, to permit expression of kanMX.
The outgrowth can be done either in liquid or on plates, for anywhere from 2
hours to overnight. Some strains require a longer outgrowth time than others.
One convenient thing to do is simply plate your transformation on YPD
plates, let them grow overnight at 30 degrees, then replica-plate to YPD
containing 200 mg/liter G418. You can also pre-determine the minimum
outgrowth time that your strain requires, in order to save some time. See
Wach, A. (1996) Yeast 12, 259-265.
Ed Davis, Ph.D.
ABL-Basic Research Program
P.O. Box B
Frederick, MD 21702-1201
Tel (301) 846-1564
FAX (301) 846-6911
reply to: davisenospam at ncifcrf.govnospam (remove "nospam" from address)
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