lichten at HELIX.NIH.GOV
Thu Jun 29 12:21:08 EST 1995
>We are currently engaged in various yeast two-hybrid library screens,
>using the LiAc transformation protocol. However it would save a lot of
>effort if we could increase our transformation efficiencies beyond
>10,000 per ug. Has anyone here used electroporation to transform yeast?
Yes. A method is in the Guthrie/Fink methods in enzymology yeast volume.
Our version follows:
Electroporation of yeast
1. Grow overnight culture in YEPD. All subsequent steps should be at 4=
2. Spin out cells, wash 3 times in 1M sorbitol. Pellet cells, decant
as much supernatant as possible, and resuspend by votexing pellet. Final
yield is approx 30 ul. cells/ml original overnight.
3. Transfer 40ul cell suspension to a cold eppendorf tube. Add:
Integrative transformation Plasmid transformation
12.5 ug sonicated carrier 12.5 ug sonicated carrier
1-2 ug cut plasmid DNA <500 ng circular plasmid
DNA must be in water or TE. Any salt will cause arcing and cook the
sample. WHEN IN DOUBT, DESALT!
4. Add cell/DNA mix to pre-cooled 0.2 cm electroporation cell. DRY
CUVETTE IMMEDIATELY BEFORE ELECTROPORATION.
5. Electroporate at 1.5 kV (kilovolts), 200 ohms, 25 microFarads.
6. IMMEDIATELY add 0.4 ml ice cold 1M sorbitol.
7. Plate cells onto selective media made with 1M sorbitol. If you
dilute cells, do so in 1M sorbitol.
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lichten at helix.nih.gov
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