Peter Novick Lab, Department of Cell Biology Yale University School of Medicine
http://info.med.yale.edu/cellbio/Novick/Second/Protocols/Lithium.pdf
A. Preparing competent yeast cells
1. Grow a 50ml overnight culture in YPD. (47.5ml YP 2.5ml 40% gluc)
Inoculate at 5:00 p.m. and shake at appropriate temperature.
2. Next morning read the OD600 against a YP blank.
3. Transfer culture to a 50 ml sterile tube and spin 4 min in a tabletop
centrifuge at room temp.
4. Pour off supernatant, resuspend in 10 ml sterile water, and spin 2 min, then pour off
supernatant.
5. Resuspend in 10 ml LITE (0.1M Li acetate, 10mM Tris, 1mM EDTA)
Prepared by:
1.5 ml 1M Lithium acetate
0.15 ml 1M Tris pH 8.0
0.03 ml 0.5M EDTA pH 8.0
add sterile water to 15 ml
6. Rock at 30oC for 1 hour.
7. Pellet 2 min, pour off supernatant and resuspend in a volume of LITE
equal to the OD600 of the original culture (for example, if A=1.0, then resuspend in 1 ml of
LITE).
8. These cells are now competent and can be stored on ice several hours.
B. Transformation of yeast cells
1. Add the following to a sterile 5 ml glass tube:
10 µl of carrier
DNA at 10 mg/ml (chick blood
DNA)
1 µgm of plasmid
DNA (dissolved in TE)
50 µl of competent yeast cells
2. Rock at 30oC for 30 min
3. Add 0.7 ml PEG-Li acetate, made as:
40% PEG (polyethylene glycol, precips
DNA onto cell surface)
0.1 M Lithium acetate
4. Rock at 30oC for 30 min.
5. Transfer to a heat block at 42oC for 2 min.
6. Plate 0.3 ml onto each of two selective plates.
7. Place plates at proper temperature (25oC)
8. Transformants will usually come up in 3-4 days.
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