Retrieval of Shuttle Plasmids from Yeast
· From: Strathern & Higgins, 1991, Methods Enzymol. 194:319-329.
1. Inoculate 4 mls of minimal selective medium in 15 ml round-bottom plastic tube with a colony
from a selective plate. Grow overnight on drum rotator at appropriate temperature.
2. Spin for 5 minutes at 2000 rpm in IEC centrifuge. Remove all supernatant. Unless otherwise
indicated, all steps are done at room temperature.
3. Resuspend cell pellet in 1 ml ddH2O. Transfer to microfuge tube. Spin 15 seconds at top speed.
4. Resuspend cell pellet in 200 µl of TENS buffer: 100 mM NaCl
10 mM Tris HCl, pH 8
1 mM EDTA
5. Quickly transfer cells to 2.0 ml tube with 0.25 g glass beads. Vortex vigorously for 1 minute.
6. Add 200 µl of phenol. Vortex vigorously for 1 minute.
7. Spin in microfuge at top speed for 5 minutes. Transfer ~250 µl to fresh microfuge tube. Do not
transfer any of the white interface; only the clear aqueous layer.
8. Extract aqueous layer with an equal volume of phenol:CHCl3 as described above. Transfer ~250 µl
to a fresh tube.
9. Add 1/10 volume of 3 M NaOAc, pH 5. Vortex. Add 2 volumes 100% EtOH. Hold at -20°C for 1