High Efficiency Transformation of Yeast Cells

Saturday, October 18, 2003

Description
This protocol uses a lithium acetate/PEG treatment to transform yeast with DNA. Plasmid DNA or linear DNA may be used

Procedure
1. Use cells from an overnight yeast culture to inoculate 300 ml of liquid YPD Medium.

2. Grow the culture until the cell density reaches 5 to 10 X 106 cells per ml (see Hint #1).

3. Centrifuge the culture in a GSA rotor at 5,000 rpm (4,000 X g) for 5 min at room temperature.

4. Remove the supernatant and resuspend the cell pellet in 10 ml of sterile ddH2O and transfer the suspension to 40 ml tubes.

5. Centrifuge the cells in a SS34 rotor at 7,000 rpm (5,900 X g) for 5 min at room temperature.

6. Remove the supernatant and resuspend the cells in 1.5 ml of sterile 1X TEL Solution.

7. Sonicate the cells for 3 min in a Branson 2200 ultrasonic waterbath (see Hint #2).

8. Incubate the cells for 1 hr at 30C with constant agitation (see Hint #3).

9. Add 100 g of Salmon Sperm DNA as a carrier and up to 5 g of the transforming DNA in a maximum volume of 20 l to microcentrifuge tubes (see Hint #4). Set up a negative control tube containing only carrier DNA.

10. Add 200 l of yeast suspension from Step 5 to each microcentrifuge tube.

11. Incubate the cells for 30 min at 30C with agitation (see Hint #3).

12. Add 1.2 ml of sterile TEL/40% PEG 4000 Solution to each tube.

13. Incubate for 30 min at 30C with agitation. (see Hint #3)

14. Heat shock the cells at 42C for 10 min (see Hint #5).

15. Centrifuge the tubes in a microcentrifuge at top speed for 5 sec.

16. Wash the cells once or twice with 0.5 ml of sterile 1X TE Buffer:

Thoroughly resuspend the cells in TE Buffer, centrifuge at top speed for 5 sec, then discard the supernatant.

17. Resuspend the final cell pellet in 1 ml of sterile 1X TE Buffer and plate 200 l of the cell suspension onto each SD selection plate containing the necessary amino acid supplements.

18. Incubate the plates at 30C (or permissive temperature) until transformants appear.

Recipes
40% Glucose 40% (w/v) D-Glucose
Autoclave to sterilize

YPD Media Make up to 950 ml with ddH2O
When cooled to 65C, add 50 ml of sterile 40% Glucose
20 g Peptone
10 g Yeast Extract
Autoclave for 30 to 45 min

SD Plates When cooled to 65C, add 50 ml of sterile 40% Glucose
1 liter of ddH2O
6.7 g Yeast Nitrogen Base (without Amino Acids, Difco)
Pour plates
Autoclave for 30 to 45 min
20 g Agar, Difco

50% PEG 4000 50% (w/v) Polyethylene Glycol MW approximately 3350-4000
Filter sterilize

TEL/40% PEG 4000 Solution (20ml) 16 ml of 50% PEG 4000
2 ml of 10X TE Buffer
2 ml of 1 M Lithium Acetate

Plasmid DNA or PCR product

Salmon Sperm DNA see protocol for Preparation of Salmon Sperm DNA for Yeast Transformations

TEL Solution Dilute with sterile ddH2O
1X TE Buffer
0.1 M Lithium Acetate

1 M Lithium Acetate Autoclave or filter-sterilize
Adjust pH to 7.5 with dilute Acetic Acid

TE Buffer (10X) Autoclave or filter-sterilize
0.1 M Tris-HCl
0.01 M EDTA
pH 7.5

Supplies

Tips
1. Two-fold to 3-fold higher efficiency is obtained by diluting to 2 million cells per ml in fresh YPD and growing for another 2 generations.

2. This is important only to obtain the highest transformation efficiency.

3. For temperature-sensitive strains, incubate at the permissive temperature for the strain.

4. The quality of the Salmon Sperm carrier DNA is the single most important factor for high efficiency transformations.

5. Incubation at 42C is fine for most temperature-sensitive mutants.

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High Efficiency Transformation of Yeast Cells