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Forsburg Lab Protocols: Transformation

Three methods are provided: electroporation, lithium acetate, and spheroplasting.
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Electroporation

This procedure stiches together observations from several sources, including different methods papers, and the documentation that comes with different electroporators.
1. Grow cells to a density of 1 x 107/ml (OD595 ca. 0.5) in minimal medium. Transformation frequency is not harmed by growth until early stationary phase ( OD595 = 1.5).
2. Harvest cells by spinning at 3000 rpm for 5 minutes at 20*C . Wash once in ice-cold water and harvesting; a second time in ice-cold 1M sorbitol. It has been reported (Suga M, Hatakeyama T.(2001) Yeast 18:1015-21) that 15m incubation of these cells in the presence of DTT at 25mM increases electrocompetence.
3. The final resuspension is in ice-cold 1M sorbitol at a density of 1 - 5 x 109 / ml.
4. 40 ul of the cell suspension are added to chilled eppendorfs containing the DNA for transformation (100 ng) and incubated on ice for 5 minutes.
5. The electroporator is set as follows (Use your manufacturer’s suggested settings if in doubt)
  • 1.5kV, 200 ohms, 25uF (Biorad)
  • 1.5 kV, 132 ohms, 40 uF (Jensen/Flowgen).

6. Cells and DNA are transferred to a pre-chilled cuvette and pulsed; 0.9 ml of ice-cold 1M sorbitol is then immediately added to the cuvette; the cell suspension is then returned to the eppendorf and placed on ice while other electroporations are carried out. You can reduce the amount of added sorbitol to 0.3ml with no significant effect.
7. Cells are plated as soon as possible onto minimal selective medium. You do NOT need to put sorbitol into the agar; in fact, if you do so you will retard growth. Transformants appear in 4 - 6 days at 32*C

Notes
Transformation efficiencies of between 105 to 106 have been reported, but more commonly we observe around 103 to 104 per microgram of DNA. It is possible that a recovery period after electroporation might improve the frequency of viable transformants. It has been suggested in budding yeast that nonsense suppressor strains are inherently osmotically unstable in electroporation; this may be the case in pombe, because anecdotal evidence suggest that sup3-5 markers do not always electroporate as efficiently. However, there is usually not a problem in getting sufficient colonies if you are simply putting a plasmid into a strain.

Pretreatment with DTT may enhance efficiency: Suga M and Hatakeyama T. (2001) High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation. Yeast 18: 1015-1021.

Electrocompetent cells can be pre-made and stored frozen: Suga M, Hatakeyama T. 2003 High-efficiency electroporation by freezing intact yeast cells with addition of calcium. Curr Genet. 43:206-211.
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Lithium Acetate

This is the "classic" high-efficiency version derived from Okazaki, K., Okazaki, N., Kume, K., Jinno, S., Tanaka, K. and Okayama, H. (1990). High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe. Nucl. Acids Res. 18:6485-6489. Immediately following is a rapid, small scale version.

1. Grow 150 ml culture in minimal medium to a density of 0.5 -1 x 107 cells/ml ( OD595 = 0.2-0.5). Media with low glucose, or MB media (see reference by Okazaki), in which the cells are less happy, may increase transformation efficiency.
2. Harvest the cells at 3000 rpm for 5 minutes at room temperature.
3. Wash cells in 40 ml of sterile water and spin them down as before.
4. Resuspend the cells at 1 x 109 cells/ml in 0.1 M lithium acatate (adjusted to pH 4.9 with acetic acid) and dispense 100 ul aliquots into eppendorf tubes. Incubate at 30*C (25*C for ts mutants) for 60 - 120 minutes. Cells will sediment at this stage.
5. Add 1 ug of plasmid DNA in 15 ul TE (pH 7.5) to each tube and mix by gentle vortexing, completely resuspending cells sedimented during the incubation. Do not allow the tubes to cool down at this stage. Add 290 µl of 50 % (w/v) PEG 4000 prewarmed at 30*C (25*C for ts mutants). Mix by gentle vortexing and incubate at 30*C (25*C for ts mutants) for 60 minutes.
6. Heat shock at 43*C for 15 minutes. Cool the tubes to room temperature for 10 minutes.
7. Centrifuge at 5000 rpm for 2 minutes in an eppendorf centrifuge. Carefully remove the supernatant by aspiration.
8. Resuspend the cells in 1 ml of 1/2 YE broth by pipetting up and down with a pipetman P1000.
9. Transfer the suspension to a 50 ml flask and dilute with 9 ml of 1/2 YE. Incubate with shaking at 32*C (25*C for ts mutants) for 60 minutes or longer.
11. Plate aliquots of less than 0.3 ml onto minimal plates. If necessary, centrifuge the cells at this stage and resuspend in 1ml of media to spread more cells on a plate.

Notes:
Expected transformation frequency is 104 to 105 per microgram of DNA.

Rapid lithium acetate

From Kanter-Smoler G et al. (1994) Improved method for rapid transformation of intact Schizosaccharomyces pombe cells. Biotechniques. Biotechniques 16:798-800.

1. Grow 10mL of culture in EMM low glucose to a density of 0.5 -1 x 107 cells/ml (OD600= 0.2-0.5)
2. Wash once in sterile water and resuspend in 1ml sterile water. Transfer to a sterile 1.5ml Eppendorf tube and pellet cells.
3. Wash once with 0.2ml of LiAc/EDTA and resuspend in 50ul of LiAc/EDTA.
4. Add DNA in up to 30ul of water or TE, and add 300ul of LiAC PEG solution.
5. Incubate with agitation at 30*C for 30 minutes.
6. Heat shock at 42*C for 15 minutes (without agitation)
7. Pellet by a brief centrifugation. Resuspend in 1ml of TE and plate 200ul aliquots on selective plates. Notes: expect about 2 x 104 transformants per microgram.
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Protoplast procedure

This method may be used for cells that are refractory to other methods. You can also force protoplasted cells to fuse in order to make diploids between non-mating strains.

1. Grow 200 ml culture to OD595 of 0.2-0.5 ( up to 1 x 107 cells/ml) in minimal medium containing 0.5% glucose and supplements.
2. Harvest cells at low speed (eg, 2-5k) decant supernatant and resuspend the pellet in 10 ml of:
    20mM Citrate/ phosphate pH 5.6 ( 2.82g/l Na2HPO4, 4.2 g/l citric acid)
    40mM EDTA pH 8.0
transfer to 50 ml plastic centrifuge tube
3. Harvest cells and resuspend each tube in 5 ml of:
    50 mM Citrate/ Phosphate pH 5.6 ( 7.1 g/l Na2HPO4, 11.5 g/l citric acid)
    1.2M Sorbitol. Adjust to pH 5.6 with 5M NaOH.

Add 25 mg NovoZymTM 234 immediately before use. Incubate at 37*C for 15-30 minutes (check under the microscope for cells to round up) until spheroplasts have formed.
Using different enzymes (e.g., Sigma's lysing enzyme) changes the shape of the cells when protoplasting. This is because different enzymes break different sugar linkages. Depending upon what linkages you break, sometimes the cells get round; sometimes they stay rodlike; sometimes they ooze out of their jackets like bright little refractile balloons, and leave ghosts behind. You can check protoplasting efficiency for any morphology by taking a small aliquot ( a few ul) and drop them on a slide with a large drop of 0.5% - 1% SDS. Protoplasted pombe will lyse in these conditions.
4. Add 35 ml of:
    10mM Tris-HCl pH 7.6
    1.2M sorbitol
and divide between 2-4 tubes ( there should be no more than 3 x 108 spheroplasts / tube). Spin gently at 2000 rpm for 5 minutes.
5. Wash twice more in 20 ml each time resuspending gently in 1ml first. At the last resuspension take a sample and count the number of protoplasts with a haemocytometer.
6. Resuspend at 2-5 x 108 protoplasts/ ml in:
    10mM Tris HCl pH 7.6
    10mM CaCl2
    1.2M Sorbitol
and combine the tubes.
7. Using 100 ul protoplast/ transformation, add 1-10 ug of transforming plasmid in up to 1/10 total volume. Incubate at room temperature for 15 minutes.
8. Add 1ml of:
    10mM Tris-HCl pH 7.6
    10mM CaCl2
    20% PEG 4000
and incubate at room temperature for 15 minutes.
9. Spin at 2000 rpm for 5 minutes, drain well and resuspend the protoplasts in 0.2-0.5 ml of:

    10mM CaCl2
    1.2M sorbitol
    0.5mg/ml Yeast extract
    5ug/ml supplements ( leu, ura, ade, his)
Incubate at 30*C for 30-60 minutes.
10. Plate out 0.2 ml aliquots onto well dried minimal sorbitol plates. Transformants appear in 2-5 days at 29-32*C. Transformation frequency is about 1 x 104 to 5 x 104 transformants/ ug DNA.

Notes: Protoplasts can be aliquotted out, stored at -70*C in 10 mM Tris-HCl pH 7.6, 10mM CaCl2, 1.2 M sorbitol (same as step 6) and used for at least 2 months. The frequency of transformation is 1 x103 transformants/ug DNA for protoplasts stored in this way.