Modified Yeast Transformation

互联网2013-09-06

698

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 of OD600 = 1 to 2 the next morning.

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 inoculum and use the one at the best OD in the morning.

Pellet the cells in a GSA rotor (5000 rpm, 5 min., RT).

Discard the supernatant and resuspend the pellet in 10 ml T.E. Transfer the cells to a 50 ml centrifuge tube.

Pellet the cells in an SS34 rotor (7000 rpm, 5 min., RT).

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.

Pellet cells in microfuge at 7000rpm, discard sup. and resuspend in 1.5mL TE/LiAC.

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.

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. up to 1.5hrs (longer is fine if maximum efficiency is not required; 1.5hrs is optimum) with shaking.

Add dimethyl sulfoxide to 10% (70uL). Mix gently.

Heat pulse for 15 min. in a 42°C waterbath.

Pellet cells in microfuge at 7000rpm, pour off most of sup and plate on appropriate medium.

Incubate the plates at 30°C until colonies appear, usually 2-3 days.

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