Okayama Competent Cells
From Gene 96:23-28 (1990)

Inoculate two very clean Fernbach flasks (no soap, bleached, and autoclaved) autoclaved with 500 mL SOB.
Use a few colonies, but offset the amount of innoculum in the two flasks so that one will be ready when you are.
Grow at RT with vigorous shaking until OD 600 nm is 0.6 (20-36 hrs) . Place flask in ice-water bath 10 min
Split cells into 2 × 250 mL pre-chilled conical tubes. Spin at 3750 rpm, 10 min, 4°C .
Gently resuspend each pellet in 80 mL ice-cold TB using 25 mL plastic pipet and pool (Total volume 160 mL).
Ice for 10 min
Spin as above, gently resuspend in each pellet in 80 mL ice-cold TB with plastic pipet
Ice for 10 min and spin down
Resuspend in 20 mL TB/pellet. With continuous swirling, add 1.4 mL/pellet fresh tissue culture-grade DMSO.
Ice for 10 min. Use this time to aliquot 100/200 μL into 1.5 mL Eppie tubes pre-chilled on ice
Freeze in liquid nitrogen. Store at -80°C. Do not refreeze once thawed

To transform
In pre-chilled 1.5 mL Eppie tube, combine 5 μL ligation +100 μL cells; swirl to mix
Ice for 20-30 min, heat shock at precisely 42°C for 90 sec, ice for 2 min
For AmpR, plate directly. For KanR, grow out the cells for 1 hour at 37°C in 1 mL LB with no drug.
Spin to harvest, remove sup but leave ?100 μL media, mix cells into this volume, and plate
Want 107-108 colonies/γ DNA. Include No DNA for test as well, 1/100 ng DNA, and 3 × 1:10 dilutions for test

SOB
(1 L, split into two flasks)
20 g bacto-tryptone
5 g yeast extract
0.5 g NaCl
10 mL 250 mM KCl
pH to 7.0 with 5 M NaOH (expect to add 0.2 mL)
autoclave, cool, then remember to add 5 mL of a 2 M MgCl2 stock that has been previously sterilized

TB
(need 400 mL per 1 L SOB culture) 1.2 g HEPES
9.3 g KCl (200 mM KCl final)
1.1 g CaCl2•2 H2O (can use other CaCl2 but adjust molarity accordingly)
Adjust volume to near but still less than 500 mL
pH to 6.7 with 10 M KOH
add 5.4 g MnCl2•4 H2O
make to 500 mL, filter to sterilize, store chilled