Colony Plasmid Rescue
Protocol adapted by Julia Sidorova and Linda Breeden from Hoffman and Winston, Gene, 1987, Vol. 57: 267-272.
- Start with a fresh plate of yeast, with large colonies grown for 3-4 days.
- 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).
- 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.
- Add 10 µl phenol and 10 µl chloroform (or 20 µl phenol:chloroform) to each eppendorf tube.
- Add 1/3 eppendorf cap (0.65 ml tube) of glass beads.
- Vortex 5 min. at speed of 4-5 in multihead vortexer at room temperature.
- Spin for 5 min. at maximum speed in a microcentrifuge.
- Thaw transformation competent E. coli cells on ice.
- 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
- Mix and incubate on ice for 30 min.
- 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)
- Shake gently at 37 deg C for 1 hr. (If using microfuge tubes, rotate at 37°C in roller drum)
- Pellet cells for 5 min. at 3 krpm in table top centrifuge.
- Pour off the supernatant, resuspend the pellet in the residual liquid, and plate the entire suspension on selective medium.