Help - yeast plasmid rapid miniprep needed!
espinoza at cgl.ucsf.edu
Tue Apr 16 20:07:43 EST 1996
stephan.witte at uni-konstanz.de (Stephan Witte) writes:
> 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 :-()
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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