Preparation of competent bacteria

Get prepared

TB buffer:
Pipes (10 mM final)        1,51 g
MnCl2 (55 mM final)        5,44 g
CaCl2 (15 mM final)        1,1 g
KCl (250 mM final)        9,32 g
H2O:                qsp 500 ml
Mettre tout sauf MnCl2, agiter et ajuster le pH à 6,7 avec KOH. Puis dissoudre MnCl2. Filtrer (0.22 μm) et stocker à 4°C.

2M Mg2+ solution: CAUTION: If starting from anhydric powders, these are extremely reactive to water, handle with care and protection
MgCl2 (1M final)    20,33 g
MgSO4 (1M final)    24,65 g
H2O:            qsp 100 ml

2M glucose: 36,03 g of glucose per 100 ml, filter sterilize.

SOB/SOC medium:
To 950 ml of H2O, add:
Tryptone        20 g
Yeast extract        5 g
NaCl            0,5 g
Shake/heat until dissolved. Add 10 ml of a 250 mM KCl solution (made by 1.86 g of KCl in 100 ml water). Adjust pH of the medium to 7.0 with  5N NaOH (~0.2 ml). Adjust volume to 1 liter with water. Autoclave.
For SOB: Just before use, add 10 ml of 2M Mg2+ solution per liter.
For SOC: Just before use, add 10 ml of 2M Mg2+ solution and 10ml of 2M glucose per liter.

Let's do it:

Day1: From fresh DH5α colonies on LB plate, inoculate a 50 ml starter of SOB+ Mg2+ , overnight 37°C

Day2: Set incubator at 18°C. At about 2:00 pm, inoculate 250 ml of SOB+ Mg2+ with 500μl of starter culture, in a 2-liters flask. Incubate in shaker at 200 rpm, 18°C.

Day4 (and not 3): Cool down to 4°C centrifuge rotor for 50ml tubes. Put TB on ice. Put sterile eppendorf tubes at -20°C.
At DO600 = 0.6 (around 11:00 am), place the flask on ice > 10 min. Spin at 2500 x g in 50 ml tubes, for 10 min at 4°C. Resuspend pellet in 80 ml ice-cold TB. Incubate on ice for 10 min, centrifuge as above. Gently resuspend pellet in 20ml ice-cold TB, and add 1.4 ml DMSO while swirling. Incubate on ice 10 min. Dispense the cell suspension in 200 μl aliquots in cold 1.5 ml tubes, immediately freeze in liquid N2. Store at -80°C. Test efficiency with 1 pg of plasmid. Should be > 108 colonnies per μg. (ideally ~ 109).

Transformation protocol

Set 42°C and 37°C waterbaths
Thaw cells on ice. Incubate with plasmid 30 minutes on ice. Put 30sec at 42°C, then back on ice. Add 800 μl of SOC, incubate 1h at 37°C.
Dry plates in incubator
Spin 2min at 3000 rpm, discard 600μl from top and plate the rest.

Dernière mise à jour : ( 26-11-2007 )