Help - yeast plasmid rapid miniprep needed!

Hernan Espinoza espinoza at cgl.ucsf.edu
Tue Apr 16 20:07:43 EST 1996


stephan.witte at uni-konstanz.de (Stephan Witte) writes:

>Hi netters!

>					After a two hybrid/interaction trap screen I have to isolate plasmids
>from my positive clones.
>I didn«t succeed with a zymolase protocol, I suppose the enzyme does not
>lyse the cells.
>Are there other reliable protocols, maybe something like alkaline
>lysis...... (not much YEAST experience :-()

Howdy, 

	This is the protocol we use for plasmid rescue (from S. cerevisiae)
in our lab (I'm afraid I can't give proper credit beyond that, sorry)

	1) Scrape a large toothpick of cells from a plate, or 
spin down 0.5 ml culture. Wash with 1ml sterile water.

2)Spin down and resuspend cells in 200 µl lysis buffer.

		Lysis Buffer = 	4% Triton X-100
			       50  mM Tris, pH 8.0
		                2.5 M LiCl
			       62.5mM EDTA, pH 8.0

3) Add 200 microlitres Phenol:Chloroform

4) Add 0.2g acid-washed glass beads and bead beat for 1 min (from Biospec
Products) or ~3min on a vortexer

5) Spin for 2 minutes in a microfuge and transfer supernatant
to a new tube

6) Add 2.5 volumes ethanol

7) Spin at 4C for 10 min

8) Wash the pellet with 70% Ethanol

9) Dissolve the pellet in 30µL TE

10) Use 3µl to transform E. coli by *electroporation* (We have had no
success with other transformation methods)

Good Luck! -Hernan



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