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