Yeast transformation using lithium acetate (rapid method)

 

Yeast transformation using lithium acetate (rapid method)

Steve Hahn

Last Modified December 1997

This method works well when transforming with a plasmid and is rapid. (for highest efficiency transformation, see DMSO transformation method)

Grow 100 ml cells overnight in YPD to an A600 of ~1.0-2.0. (A small blob of cells from a plate inoculated into 100 ml YPD and grown for 14 hr at 30^o is usually fine).

Harvest cells (100 ml) by centrifugation (5K rpm for 5 min in GSA). If cell density is greater than 1.0, use correspondingly less cells.

Resuspend cells in 5 ml TE pH 7.5 and transfer to 15 ml screwcap tube. Spin 2 min in clinical centrifuge.

Resuspend cell pellet in 5 ml TE + 0.1 M lithium acetate. Spin 2 min in clinical centrifuge.

Resuspend cell pellet in 2 ml TE + 0.1 M lithium acetate. Incubate on tube roller at 30o for 1 hr (can be incubated for up to four hours with no ill effects).

Boil high molecular weight salmon sperm DNA for 3-5 min. Rapidly chill on ice (this separates DNA strands, making it single stranded).

In a sterile eppendorf tube, add:

1. 10 microliters (10 mg/ml) single stranded high molecular weight salmon sperm DNA

2. Plasmid DNA for transformation (1 microgram should give >1000 transformants; 1-2 microliters of mini-prep DNA works well).

3. 0.2 ml cells treated with LiOAc from above.

4. 1 ml 40% PEG 4000, 1XTE pH 7.5, 0.1 M Lithium acetate.

Mix by vortexing and incubate at 30 degrees for 30 min.

Heat shock cells for 15 min. at 42 degrees.

Spin 5 sec in microfuge and remove supernatant by dumping off.

Resuspend cell pellet in 1 ml TE using a sterile pipetman tip and vortexing.

Spin in microfuge 5 sec. Remove supernatant by dumping off.

Resuspend cell pellet in 0.5 ml TE by vortexing.

Plate 0.2 ml to selective plates.