Yeast Transformation

I. Start O/N culture in YPD, 5mls per transformation

II. Prepare cells (10ml example)
    1. Start grow cells by diluting O/N cultured cells to 0.1OD/ml

    2. Grow until OD= 0.5~1.0 (1OD unit = 1 X 10⁷ cells/ml)

    3. Spin cells 3000 rpm/5min
    4. Wash with 10ml chilled H2O
    5. Wash with 1ml 1X TE/LiAc (fresh prep)

    6. Resuspend ~75ul 1X TE/LiAc (final volume will be ~100ul)

III. Transformation
     1. Use 50ul per transformation
     2. Mix 5ul s.s. carrier DNA (50ug) and 2ul minipreped plasmid (~1ug)
     3. Mix DNA and yeast

     4. Add 300ul 40% PEG/TE/LiAc (fresh prep)
     5. Rotate RT for 30min
     6. Heat Shock for 15min at 42C
     7. Spin down 4000rpm/4min
     8. Wash with 500ul TE buffer

     9. Resuspend with 500ul TE buffer

     10. Plate ~200ul on appropriate plate

     11. Grow 30C

IV. Solution

      1. 10X TE
              0.1M Tris
              0.01M EDTA pH8.0
      

      2. 10X LiAc
              1M LiAc pH 7.5 with dilute HOAc

      3. 50% PEG 3350