Yeast Transformation
- inoculate 5-10 ml culture with single colony and grow ON
- measure OD600nm and dilute to OD600nm = 0.3 in 50 ml
- grow for 2-4 hrs
- spin, wash cells with 25 ml H2O
- spin, resuspend in 2 ml 1 x LiAc/ 0.5 x TE
- incubate at RT for 10 min
- combine in tube: 100 µl cells, 10 µl salmon sperm DNA (10 mg/ml), up to 15 µl DNA to be transformed
- add 700 µl 1 x LiAc / 1 x TE / 40 % PEG mix
- incubate for 30 min at 30 °C (shaking or not shaking)
- add 85 µl DMSO
- heat shock at 42 °C for 7 min
- spin, resuspend cells in 1 ml 1 x TE
- spin, resuspend cells in 0.5 ml 1 x TE
- plate 100 - 200 µl / plate and incubate at 30 °C
Buffers:
10 x TE: 100 mM Tris pH 7.5; 10 mM
EDTA.
10 x LiAc: 1 M LiAc in H2O;
sterile filter.
50 % PEG 3350: 50 % PEG 3350 in H2O;
sterile filter.
