Team:Goettingen/protocol Plasmid Tran

Preparation of E.coli competence cell

E.coli transformation

- Put 1μl of circular plasmid or all of a ligation reaction of plasmid DNA in a 1.5 mL tube. Gently add ~100 μl of competent cells.
- Incubate for 30 min on ice.
- Heat shock for 2 min @ 42°C. Put back on ice.
- Add 900 μl of LB to tubes. Incubate @ 37°C for 30 min.
- Plate different concentrations (e.g. 50, 100, rest μL) of the cells on LB + antibiotics plates. Incubate them @ 37°C overnight.

Plasmid Isolation from Yeast

1. Collect cells by centrifugation ( room temperature, 4000 rpm, 5 min.) and discard the supernatant.
2. Resuspend the pellet in 250 µl of buffer P1.
3. Add ca. 0.4 g glass beads and break the cells by vortexing vigorously for 5 min.
4. Add 250 µl of buffer P2 and mix immediately.
5. Add 350 µl of buffer N3 and mix thoroughly.
6. Harvest the supernatant (13,000 rpm, 10 min.), transfer it to a new 1.5 ml E-cup.
7. Add 750 µl ice cold isopropanol and incubate 20 min. at 20°C.
8. Harvest the plasmid-DNA (13,000 rpm, 10 min.), discard the supernatant.
9. Wash the plasmid-DNA: Add 500 µl ice cold 70% EtOH and invert 4-6 time and centrifuge (13,000 rpm, 10 min.).
10. Discard supernatant, dry the DNA pellet and resolve the plasmid-DNA in 50 µl sterile distilled water.
11. Measure DNA concentration with Nanodrop.

Yeast transformation

- Pick a colony into 5 ml YPAD medium (pH 5.8) and incubate overnight at 30°C with shaking. Until OD600 >1.5.
- Sub-culture 1 ml into 50 ml YPAD and grow at 30°C with shaking for 3.5-4h. Until OD600 0.6-1.2.
- Collect cells by low speed centrifugation (4000 rpm, 5 min, room temperature)
- Discard the medium and resuspend the cells by vortexing in 25 ml dH2O.
- Respin cells (4000 rpm, 5 min, room temperature), discard supernatant and resuspend in 1 ml sterile dH2O.
- Transfer to a 1.5 mL tube and respin at top speed for 10 sec to pellet cells.
- Resuspend in 550 μl 100 mM LiAc pH7.5 and transfer 50 μl aliquots to 11 sterile microcentrifuge tubes.
- Centrifuge for 10 s at top speed to pellet cells, and remove the supernatant.
- To each tube add, in order: 240 μl 50% PEG 4000, 36 μl 1 M LiAc, 10 μl single-stranded DNA (Salmon sperm, Invitrogen).
- 0.5-1 μl plasmid DNA (250-500 ng of each plasmid)
- Resuspend and mix thoroughly by pipetting or vigorous vortexing. Incubate at 42°C for 25 - 45 min.
- Centrifuge cells at low speed (4000 rpm, 10 sec), remove medium and resuspend cell pellet in 1 ml YPAD for 1 h.
- Centrifuge cells at low speed (4000 rpm, 1 min) remove medium and resuspend cell pellet in 200 μl sterile dH2O.
- Spread aliquots onto SC dropout medium (spread gently with spreading bar or using sterile glass beads). Allow to air-dry and incubate at 30°C for 2-3 days for colonies to grow.