Electropor. in Yeast

Tomas Drgon tomasd at bdg10.niddk.nih.gov
Wed Sep 20 08:55:53 EST 1995


In article <cve-1509951217500001 at 193.174.107.194>, cve at ifm.mh-hannover.de
(Christian Velten) wrote:

> Hi everybody,
> 
> does anyone know a good source for the electroporation of S. cerevisiae
method.
> 
> Thanks,
> 
> Christian

Hi.
A protocol usually comes with the cuvettes. This one came with the BRL ones:

1. Grow cells in YEDP (2% pep, 1% YE, 2% gluc.) 50 ml. Harvest in log
phase (5x10^7 cells/ml).

2. Centrifuge at 2000 g 5 min 4oC. Wash cells 2x by resuspending in 25 ml
of cold sterile water. (The goal is to decrease the conductivity)

3. Resuspend cells in 250 ul of cold sterile water. Store on ice over
short time. Some people even claim you can freeze it as E.coli (never
tried).

4. Use 20 ul for 1 electroporation. Add 1 ug DNA and hold on ice for 15 min.

5. Suspend all 21 ul between the bosses of the electroporation cuvette.

6. Electroporator setting: 400 V, 10 uF and low ohmes.

7. Remove cell suspension from the cuvette, add 79 ul sterile water, and
plate the whole volume on a selective or whatever plate.


rem:  some people recommend 1M sorbitol instead of water from step 3 and
also in the plates.


Regards

Tomas Drgon

-- 
Vidno jak psoj kulky.
(Old Slovak proverb)



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