Efficient Transformation of Yeast - LiAc Method
互联网
Efficient Transformation of Yeast - LiAc Method
Different strains transform at different rates with different methods. Some strains transform much better using the electroporation method. Other ones are transformed with a very high efficiency using the following protocol:
- 10xLiAc: 1M LiAc pH 7.5 (adjust with HAc)
- 10xTE: 0.1M Tris-Cl (pH 7.5), 10mM EDTA
- 50% PEG 50% PEG 4000 in water (autocalve ONCE only)
- PEG/LiAc sol. 8ml 50% PEG, 1ml 10xTE, 1ml 10xLiAc,
-
LiAc sol. 1ml 10xLiAc, 1ml 10xTE, 8ml H2 O (water)
Transfer enough of this culture to a 300ml YPDA medium in a 1L-flask to produce an inital OD of 0.2. Incubate with shaking (250rpm) for 3hr. Centrifuge cells at 1000g for 5min at RT. Discard the supernatant and resuspend the cell pellet in 50ml H2 O. Centrifuge the cells again (same conditions as above), discard the supernatant and resuspend the pellet in 1.5ml sterile LiAc sol.
Prepare PEG/LiAc fresh. Set up the required number of eppendorf tubes and add 100µL of cells. Add 0.1µg of each type of plasmid, together with 100µg of salmon sperm DNA. Mix.
Add 0.6ml sterile PEG/LiAc sol. to each tube and vortex.
Incubate at 30°C for 30 min with shaking (250rpm). Add 70µL of 100% DMSO (=10% final conc.) and mix gently.
Heat shock for 15 min at 42°C. Chill cells on ice and pellet in a centrifuge for 5 sec at 14.000rpm. Remove the supernatant and resuspend cells in 0.5mL of TE buffer.
Spread 100µL of this mixture onto each standard size plate containing the appropriate selection medium. Incubate plates for 3-4 days until colonies appear.
