Competent cells

Competent cells (RbCl2)

10ml colture ON 37°C (no antibiotics)

put 10ml into 500 ml LB 37°C (no antibiotics)

Check every 30 min OD 550 up to 0,5 OD

Put in Falcon 50 ml and spin 4000rpm 15 min 4°C

Resuspend pellets in 50 ml total TFBI and put on ice 1h.

Spin 4000 rpm 5min and discard sup. Resuspend pellet in 20 ml TFBII. Put on ice 30min. Freeze aliquotes with liquid nitrogen and stock -80°C.

TFBI: 30mM PotassiumAcetate (1ml 3M for 100ml total); 50mM Manganese Cloride (25 ml 200mM for 100ml tot); 100mM RbCl2 (10ml 1M for 100ml tot; 1,21gr/10ml H2O); 10mM CaCl2 (1ml 1M for 100 ml tot); 15% glycerol (15 gr for 100 ml tot); pH=5,8 with acetic acid. Filter final solution. Store 4°C.

TFBII: 10mM MOPS 0,2 ml 1M for 20 ml tot); 75 mM CaCl2 (1,5ml 1M for 20 ml tot); 10mM RbCl2 0,2ml 1M for 20 ml tot); 15% glycerol (3 gr for 20ml tot). Filter final solution and store 4°C.

Competent cells (CaCl2)

Inocule 1ml/100 LB of a O/N culture (3-4 ml LB) and let it grow till 0.4-0.5 OD600

Transfer in sterile 50ml falcon and keep on ice 10’ to stop growth

Spin at 3500rpm (2100xg) at 4°C 10’, remove all the super

Resuspend (keeping on ice) in 10ml 100mM CaCl2- on ice 30’

Spin at 3500rpm (2100xg) at 4°C 10’, remove all the super

Resuspend (keeping on ice) in 10ml 100mM CaCl2 + 20% glycerol

Aliquot keeping cold (cold room) in pre-chilled Eppendorf

Store at -80°C

NB: KEEP EVERYTHING ICE COLD AND PRE-CHILL SOL’N AND TUBES