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1. Inoculate cells from an overnight culture into 50 ml YEPD and incubate at 30¡C with
shaking. Typically, add 0.1 to 0.2 ml saturated culture in the evening to get an OD6oo=1 to 2 the next morning.
2. Transfer enough of this culture to 300 ml YEPD to produce an OD6oo=0.2 (ca. 20
mls.). Incubate at 30¡C to an OD600 of 6.5 to 8.5. I often grow three cultures overnight starting with different amounts of inoculumand use the one at the best stage in the morning.
3. Pellet the cells in a GSA rotor (5000 rpm, 5 min., RT).
4. Discard the supernatant and resuspend the pellet in 10 ml T.E. Transfer the cells to a
50 ml centrifuge tube.
5. Pellet the cells in an SS34 rotor (7000 rpm, 5 min., RT).
6. Discard the supernatant and resuspend the pellet in 10 ml sterile 1 X TE/LiAC (made fresh from stocks of 10 X TE [0.1 M Tris-HCI, 0.01 M EDTA, pH 7.5] and 10X LiAC [1 M lithium acetate adjusted to pH 7.5 with dilute acetic acid]. Repeat.
7. Pellet cells in microfuge at 7000rpm, discard sup. and resuspend in 1.5ul TE/LiAC.
8. Add 100ul cells to the plasmid DNAs (1-5 ug) and single-stranded carrier DNA
(20ug) in a microfuge tube. The carrier DNA is produced by dissolving salmon sperm DNA in TE, sonicating it to reduce its viscosity, extracting it with phenol/chloroform, and precipitating it with ethanol. The DNA is resuspended at a concentration of 10 mg/ml in TE, placed in a boiling water bath for 20 min., and immediately cooled on ice. The carrier DNA solution can be stored in aliquots at-20¡C.
19. Add 700ul ml sterile PEG solution (40 % PEG, 1 X TE, 1 XLiAC, made fresh from stocks of 50 % PEG 4000, 10 X TE, 10 X LiAC) to each tube and incubate at 30¡C for 45min. with shaking.
11. Add dimethyl sulfoxide to 10%. Mix gently.
12. Heat pulse for 15 min. in a 42¡C waterbath.
13. Pellet cells in microfuge at 7000rpm, pour off most of sup and plate on appropriate medium.
14. Incubate the plates at 30¡C until colonies appear, usually 2-3 days.
Z buffer (per liter)
Na2HPO4.7H2O 16.1 g
(Na2HPO4 8.5 g)
NaH2PO4.H2O 5.5 g
KCl 0.75 g
MgSO4.7H2O 0.246 g
Adjust pH to 7.0
X-gal
Dissolve 5-bromo-4-chloro-3-indolyl--D-galactoside (X-gal) in N,N-dimenthylformamide at a concentration of 20 mg/ml.
2. Prepare a X-gal/Z buffer solution containing 100 ml of Z buffer, 0.27 ml of 2-mercaptoethanol, and
1.67 ml X-gal stock. To assay colonies on a 100 mm diameter dish, pipet 1.8 ml of this solution to a clean dish and soak a filter placed onto the solution. Use either Whatman 1 or VWR grade 413 paper
filters.
3. After the colonies have grown, they are picked and transferred to a filter (or place a sterile filter onto a transformation plate and orient it).
4. The filter is placed into a pool of liquid nitrogen for 10 seconds, then removed and allowed to thaw at room temperature.
5. Carefully layer the thawed filter, colony side up, onto the presoaked with X-gal/Z buffer solution. Colonies producing b-galactosidase will turn blue any time from 30 min to 30 hours.