S. pombe transformation by lithium acetate

Protocol written for transformation with pREP-series of plasmids. Provided by Blerta Xhemalce Strain: soSPBX5: h+, ade6-216, leu1-32, ura4-D18

Contents

1. Materials
2. Protocol

1. Materials

1.1 Media

YES

• 0.5% (w/v) yeast extract.
• 3.0% (w/v) glucose
• YE+225 mg/l adenine, leucine and uracil

EMM+Ade+Leu+0.2g/l thiamine plates for pREP with Ura marker or EMM+Ade+Ura+0.2g/l thiamine plates for pREP with Leu marker

1.2 Reagents

• 1M Lithium Acetate adjusted to pH 4.9 with acetic acid.
• 50% (w/v) PEG3350 in 0.1M AcLi, ph 4.9
• Sonicated salmon sperm DNA at 10µg/µl.

2. Protocol

1. Grow 100 ml of culture at 30°C O/N in YES medium to OD600 = 0.5.
2. Harvest the cells at 3000 rpm for 3 minutes at room temperature.
3. Wash cells in 20 ml of H2O and spin at 3000 rpm for 3 minutes at room temperature.
4. Wash cells in 20 ml of 0.1 M lithium acetate, pH 4.9 and spin them down as before.
5. Resuspend cells at 100 OD/ml in 0.1 M lithium acetate, pH 4.9 and dispense 100 μl aliquots into Eppendorf tubes.
6. Incubate at room temperature for 1h.
7. Add 1 μl of the sonicated salmon sperm DNA and 1µg of the pREP plasmid in 15 μl TE (pH 7.5) to each tube and mix by gentle vortexing, completely resuspending cells sedimented during the incubation.
8. Incubate at room temperature for 1h.
9. Add 290 μl of 50 % (w/v) PEG3350 and mix by gentle vortexing.
10. Incubate at room temperature for 1h.
11. Heat shock at 43°C for 15 minutes. Cool the tubes to room temperature for 10 minutes.
12. Centrifuge at 3000 rpm for 1 minute in an Eppendorf centrifuge. Carefully remove the supernatant by aspiration.
13. Resuspend the cells in 1 ml of 0.2X YES by pipetting up and down with a pipetman P1000.
14. Incubate with shaking at room temperature for 30 minutes or longer.
15. Plate aliquots of 0.25 ml onto EMM+thia plates and incubate for 3 to 5 days at 30°C.