Silver: LiOAc Transformation

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Materials

  • 50% Polyethylene glycol
    • 100 g (3350 g/mol) PEG
    • Add ddH2O to 200 ml
    • Mix with heat and filter (takes a while)
  • 10x TE/LiOAc
    • 20 ml 1 M Tris-HCl (100 mM final)
    • 4 ml 0.5 M EDTA (10 mM final)
    • 20.4 g LiOAc
    • 176 ml ddH2O
    • Filter
  • 40% PEG / 1xTE/LiOAc
    • 10 ml 10x TE/LiOAc
    • 80 ml 50% PEG
    • 10 ml ddH2O
    • Mix and filter (takes a while)
  • 10 mg/ml salmon sperm DNA (preboiled for 10 min)
  • DMSO
  • Sterile/autoclaved glass spreading beads
  • Selection plates (depends on experiment)
    • YEPD/G418 selection plates
      1. Mix the following ingredients in a 2 liter flask
        • 10 g yeast extract
        • 20 g bactopeptone
        • 20 g dextrose (glucose)
        • 20 g bactoagar
        • 1 L ddH2O
        • stir bar
      2. Autoclave on a liquid cycle for 45 min
      3. Cool on a stir plate
      4. Add 200 µl of 1 g/ml G418 just before pouring into plates
        • Final G418 concentration is 200 µg/ml


Protocol

  1. Grow cells to 1-2 x 107 cells/ml -- 10 ml per transformation
  2. Spin down cells 2 min / 2300 rpm
  3. Resuspend in ddH2O (10 ml per transformation), spin down again
  4. Resuspend in 1 ml ddH2O and spin 10 sec at 14,000 rpm in microfuge
  5. Wash twice with 1 ml 1x TE/LiOAc, spin (can be stored at 4°C for a week for plasmid transformations, not recommended for integrations), if you use immediately resuspend cells in 50 ul 1x TE/LiOAc
  6. Boil tube of 10 mg/ml salmon sperm DNA for 5 min, cool on ice
  7. For transformations add
    • 2.5 µl ssDNA
    • Transforming DNA (usually do three amounts)
    • 50 µl cells in 1x TE/LiOAc
  8. Add 300 µl of 40% PEG4000 (3350) / 1xTE/LiOAc, vortex well
  9. Incubate at 30°C on roller for 30 min (25°C for ts strains)
  10. Add 35 µl DMSO and vortex well
  11. Heat shock for 15 min at 42°C (15 min at 37°C or 25°C for ts strains)
  12. Pellet cells and resuspend in 150 µl ddH2O and spread all onto one plate (unless transforming with a plasmid, only do 1/10th the amount then), spread with sterile/autoclaved glass beads
  13. Incubate overnight at 25°C (ts strains) or 30°C on a selective media plate
    • For G418 resistance you need to let the cells recover, do either of these two options:
      1. Plate on YEPD and grow overnight then replica-plate onto YEPD/G418 the next day
      2. Inoculate YEPD liquid and let grow for 4 h at 30°C, then plate onto YEPD/G418
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