Team:Goettingen/protocol Plasmid Tran

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.

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.