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Breeden Lab, yeast transformation
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.
10 x TE: 100 mM Tris pH 7.5; 10 mM EDTA.