This protocol is very effective and simple. The fact that the cells are grown at low temperature greatly increase the OD range during which the cells will prep up at high efficiency (Inoue et al., 1990).

Chill the following: >100 ml ice-cold TB, centrifuge (GSA rotor), 500 ml centrifuge bottle, and sterile freezing tubes (eppendorf tubes w/ good caps)

-Innoculate 250 ml SOB media (2 liter flask) w/ 10-12 colonies bacteria from a fresh plate Shake well at 18°C - RT; grow bugs to A600 ~0.6 (ON-2 days). An OD of 0.6 is ideal, but anywhere from 0.2 to 1.0 will work well. [Alternatively, try diluting an overnight culture to OD=0.1 and then growing at RT for ~6 hrs.]

-Place on ice 10 minutes.

-Transfer to centrifuge bottle; spin 2500 x g , 10 min, 4°C.

-Resuspend pellet in 80 ml ice-cold TB; place on ice for 10 min.

-Spin again 2500 x g , 10 min, 4°C.

-Resuspend in 20 ml ice-cold TB.

-Add DMSO, while swirling, to final 7 % (~1.4 ml).

-Place on ice, 10 min. Dispense into freezing tubes (~0.5-1 ml ea)

-Freeze in liquid N2; transfer to -80°.

1 liter SOB: 20 g bactotryptone, 5 g bacto-yeast extract, 0.5g NaCl

Dissolve all in 950 ml ddH2O; add 10 ml 250 mM KCl; pH to 7.0 with NaOH

Adjust vol to 1 liter; Autoclave. Just before use, add 5 ml sterile 2M MgCl2 per liter

TB per liter

10 mM PIPES Na salt 3.35 g

15 mM CaCl2 (Fisher) 2.2 g

250 mM KCl (Aldrich) 18.64g

55 mM MnCl2 (Aldrich) 10.9 g

Combine PIPES, CaCl2, KCl ; pH to 6.7; add MnCl2; filter sterilize

REFERENCES

Inoue, H., Nojima, H., and Okayama, H. (1990). High efficiency transformation of Escherichia coli with plasmids. Gene 96, 23-8.