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
 * 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
 * Autoclave on a liquid cycle for 45 min
 * Cool on a stir plate
 * 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
 * 8) *2.5 µl ssDNA
 * 9) *Transforming DNA (usually do three amounts)
 * 10) *50 µl cells in 1x TE/LiOAc
 * 11) Add 300 µl of 40% PEG4000 (3350) / 1xTE/LiOAc, vortex well
 * 12) Incubate at 30°C on roller for 30 min (25°C for ts strains)
 * 13) Add 35 µl DMSO and vortex well
 * 14) Heat shock for 15 min at 42°C (15 min at 37°C or 25°C for ts strains)
 * 15) 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
 * 16) Incubate overnight at 25°C (ts strains) or 30°C on a selective media plate
 * 17) *For G418 resistance you need to let the cells recover, do either of these two options:
 * 18) *#Plate on YEPD and grow overnight then replica-plate onto YEPD/G418 the next day
 * 19) *#Inoculate YEPD liquid and let grow for 4 h at 30°C, then plate onto YEPD/G418