This is a cached page for the URL (http://www.fhcrc.org/labs/gottschling/yeast/yplas.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Yeast Protocols
Fred Hutchinson Cancer Research Center    
HOME >  Science and Research >  Gottschling Lab
 
 
 

High Efficency Yeast Plasmid Rescue

(by Trey Powers, Fields Lab)

  1. Grow yeast overnight.

  2. Spin down 1.5 ml of culture in microfuge tubes.

  3. Decant

  4. Add 250 Ál of Qiagen buffer P1 to each tube

  5. Add about 250 Ál of glass beads

  6. Beat or vortex on high for 3-5 minutes

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

  8. Add 350 Ál of buffer N3 and mix immediately, but gently

  9. Centrifuge for 10 minutes on full blast

  10. Apply supernatants to QIAprep spin column

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

  12. Wash column with 750 Ál of Buffer PE and centrifuge 45 seconds

  13. Discard flow through and spin for an additional 45 seconds

  14. Place column in a clean tube and add 50 Ál of buffer EB

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

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


Colony Plasmid Rescue

Protocol adapted by Julia Sidorova and Linda Breeden from Hoffman and Winston, Gene, 1987, Vol. 57: 267-272.
  1. Start with a fresh plate of yeast, with large colonies grown for 3-4 days.

  2. Prepare eppendorfs with 20 Ál aqueous buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0).

  3. Pick colonies from plate with a pipette tip and resuspend in the aqueous buffer.
    Note: Lab lore recommends NOT using a toothpick to pick your colony. There is something evil in toothpicks that inhibits bacterial transformations.

  4. Add 10 Ál phenol and 10 Ál chloroform (or 20 Ál phenol:chloroform) to each eppendorf tube.

  5. Add 1/3 eppendorf cap (0.65 ml tube) of glass beads.

  6. Vortex 5 min. at speed of 4-5 in multihead vortexer at room temperature.

  7. Spin for 5 min. at maximum speed in a microcentrifuge.

  8. Thaw transformation competent E. coli cells on ice.

  9. In test tubes (for microfuge tubes see below; otherwise use 14-ml snap-cap tube) on ice add:

    a) 1 Ál aqueous phase (if you experience low yield, try using more)

    b) 120 Ál competent cells

  10. Mix and incubate on ice for 30 min.

  11. Heat shock for 90 sec at 42 deg C, then immediately add 2 ml of NZY broth to each test tube. (LB will also work, though richer media is usually better)

  12. Shake gently at 37 deg C for 1 hr. (If using microfuge tubes, rotate at 37°C in roller drum)

  13. Pellet cells for 5 min. at 3 krpm in table top centrifuge.

  14. Pour off the supernatant, resuspend the pellet in the residual liquid, and plate the entire suspension on selective medium.

 
Prepared By:
Michael McMurray
(206) 667-6660
Last Modified: Jul 24, 2002


  Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, WA 98109  (206)667-5000 [ Home ][ Directory ][ News & Events ][ Donate ][ Jobs ][ Search ][ Site Map ]
  Copyright 2003 Fred Hutchinson Cancer Research Center, a non-profit organization. See Terms of Use & Privacy Policy.