Yeast Lysates for Westerns

互联网2013-09-06

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Cells are grown for 2-3 days as 1.5ml prep. under selection for the plasmid of interest. Spin cells down 2.6K for 5min.

Resuspend in 1ml 0.25m NaOH/1% 2-mercaptoethanol

Incubate 10min on ice.

Add 0.16ml 50% trichloroacetic acid

Incubate for 10min on ice.

Pellet 14K 10min.

Resuspend pellet in 1ml ice cold acetone.

Pellet 14K 10min.

Air dry pellet and resuspend in 200-500ul SDS SB and proceed as described below in section on western transfer

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