High Efficency Yeast Plasmid Rescue

Tuesday, November 18, 2003

Description
High Efficency Yeast Plasmid Rescue

Procedure
Grow yeast overnight.

Spin down 1.5 ml of culture in microfuge tubes.

Decant

Add 250 l of Qiagen buffer P1 to each tube

Add about 250 l of glass beads

Beat or vortex on high for 3-5 minutes

Add 250 l of buffer P2 and gently mix well (Do not let lysis reaction continue for more than five minutes)

Add 350 l of buffer N3 and mix immediately, but gently

Centrifuge for 10 minutes on full blast

Apply supernatants to QIAprep spin column

Centrifuge column for 45 seconds on full blast. Discard flow-though

Wash column with 750 l of Buffer PE and centrifuge 45 seconds

Discard flow through and spin for an additional 45 seconds

Place column in a clean tube and add 50 l of buffer EB

Let sit for 1 min. then spin for 1 min.

For efficent transformation into coli use 1-2 l of this prep per aliquot of E. coli using the 90sec heat shock protocol. (NOTE: using up to 20 l will increase yield of transformants)

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High Efficency Yeast Plasmid Rescue