Yeast Transformation

Yeast Transformation

• inoculate 5-10 ml culture with single colony and grow ON
• measure OD_600nm and dilute to OD_600nm = 0.3 in 50 ml
• grow for 2-4 hrs
• spin, wash cells with 25 ml H₂ O
• 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 H₂ O; sterile filter.
 50 % PEG 3350: 50 % PEG 3350 in H₂ O; sterile filter.