Yeast transformation using lithium acetate (rapid method)
Yeast transformation using lithium acetate (rapid method)
Steve Hahn , Genetic Methods
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 30o 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.
Steve Hahn , Genetic Methods
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 30o 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.
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