yeastplasmid rescue

Andrey Tsouladze tsoul at techunix.technion.ac.il
Fri Mar 18 10:13:33 EST 1994


For rescuing plasmids from yeast, we are using routinely a simple
protocol which has been developed in our lab and published in NAR (1992),
v.20, p.3790. In breaf, it goes as follows:
1. Grow 1.5 ml o/n yeast culture (cells from a plate can be used as well).
2. Pellet and resuspend in 0.1 ml of STET (8% sucrose, 50 mM Tris pH
   8.0, 50 mM EDTA, 5% Triton X-100).
3. Add 0.2 g of glass beads, vortex for 5 min.
4. Add 0.1 ml of STET, mix, boil for 3 min.
5. Cool briefly on ice, microfuge for 10 min at 4C.
6. Transfer 0.1 ml of super to 0.05 ml of 7.5 M ammonium acetate.
   Incubate at 4C for 1 hr, microfuge for 10 min at 4C.
7. Add 0.1 ml of super to 0.2 ml of ice-cold EtOH, microfuge as above.
8. Wash with 70% ethanol, dry, resuspend in 0.02 ml of water.
9. Use half the volume to transform bacteria.

A simplest calcium chloride transformation protocol gives hundreds of
transformants with a CEN plasmid, and thousands with an ARS plasmid.
There is a strain preference, and the best results we obtain with the
MC1061. Also, freshly prepared competent cells are transformed better
than those stored at -70C.

Good luck,

Andrey

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
*               Andrey Tsouladze                *                   *
*            Department of Biology              * You               *
*  Technion - Israel Institute of Technology    *   have            *
*                Haifa 32000                    *      been         *
*                  Israel                       *         warned... *
*    E-mail: tsoul at aluf.technion.ac.il          *                   *
*    E-mail: tsoul at techunix.technion.ac.il      *                   *
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