Electropor. in Yeast

BRAINSL at aol.com BRAINSL at aol.com
Mon Sep 25 07:55:11 EST 1995


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ok, In a message dated 95-09-20 11:03:09 EDT, BIOSCI-REQUEST at net.bio.net
writes:

>Subj:	RE: Electropor. in Yeast
>Date:	95-09-20 11:03:09 EDT
>From:	tomasd at bdg10.niddk.nih.gov (Tomas Drgon)
>Sender:	postman at alw.nih.gov (AMDS Postmaster)
>Resent-from:	BIOSCI-REQUEST at net.bio.net
>To:	yeast at net.bio.net
>
>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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>Date: Wed, 20 Sep 1995 13:55:53 GMT
>From: tomasd at bdg10.niddk.nih.gov (Tomas Drgon)
>Subject: RE: Electropor. in Yeast
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