High Efficency Yeast Plasmid Rescue

 

1. 1. Grow yeast overnight.
   2. Spin down 1.5 ml of culture in microfuge tubes.
   3. Decant.
   4. Add 250 µl of Qiagen buffer P1 and about 250 µl of glass beads to each tube.
   5. Beat or vortex on high for 3-5 minutes.
   6. Add 250 µl of buffer P2 and gently mix well (Do not let lysis reaction continue for more than five minutes).
   7. Add 350 µl of buffer N3 and mix immediately, but gently.
   8. Centrifuge for 10 minutes on full blast.
   9. Apply supernatants to QIAprep spin column.
   10. Centrifuge column for 45 seconds on full blast. Discard flow-though.
   11. Wash column with 750 µl of Buffer PE and centrifuge 45 seconds.
   12. Discard flow through and spin for an additional 45 seconds.
   13. Place column in a clean tube and add 50 µl of buffer EB.
   14. Let sit for 1 min. then spin for 1 min.
   15. For efficent transformation into coli use 1-2 µl of this prep per aliquot of E. coli using the 90sec heat shock protocol. (NOTE: using up to 20 µl 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 µl 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 µl phenol and 10 µl chloroform (or 20 µl 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 µl aqueous phase (if you experience low yield, try using more) b) 120 µl 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.