Frozen competent E. coli cells 【Carnegie Institution of Washington】

Inoculate a 5ml overnight of E.coli in LB+20 mM MgSO₄.

Next morning,inoculate 250 ml LB+20 mM Mg++ in a 2L flask with about 2ml overnight culture.Grow at room temp (23℃)with good aeration (250rpm)to an A600 of 0.4-0.6.Temp is important--see original ref.

Place cells 10 min on ice.Transfer to a sterile bottle and spin 3K,10',4℃.

Resuspend pellet in 80 ml cold TB (swirl cells in bottle).Leave 10’/ice.

Spin cells 3K,10',4℃.

Resuspend cells in 20 ml cold TB then add 1.5 ml DMSO.Leave 10'/ice.

Dispense into 220μl and 525μl aliquots (in cold sterile tubes)and freeze in dry ice/EtOH bath.Store -70℃.Typically,competency about 5X10⁶ cfu/ug DNA.Note,improves after freezing.Cells good for a year and counting.

To use:

To 50 or 100μl cells,add 5-50 ng DNA.Leave 30’ on ice.

Heat shock,45 sec at42℃ then chill cells on ice about 2’.

Spin down (15 sec,eppendorf fuge)and remove SN.(Removing the Manganese seems to boost efficiency about 10X)and resuspend cells in 200μl LB.

For a supercoiled plasmid,plate 1μl of cells.For a ligation,plate 20μl and the rest.

TB (transformation buffer: filter sterilize and store 4℃)