Competent cells E.coli

For DH5-alfa , BL21D ,  XL1Blue


50 ul glycerol stock cells

LB plate

50 ml sterile tubes

200 ml sterile erlenmeyer 

LB media

0.75 ml Glycerol sterile

30 ml 50 mM CaCl2 sterile 

100 sterile 1.5 ml epperdorf 


-Streak out cells from glycerol stock onto LB plate (no antibiotic) and grow overnight at 37C

-From plate, start 3 ml LB culture with 1 colony overnight, 37C, 250 rpm

-In a 200 ml erlenmeyer flask inoculate 100 ul overnight culture into 50 ml LB media.

-Grow at 37C until OD 600nm = 0.4-0.6 (3-4 hr)

-Transfer cells into falcon tube and incubate on ice 15 min  to cool them down

-Spin at 1800 g, 10 min 4C

-Remove supernatant and ressuspend in 25 ml CaCl2 gently

-Incubate 30 min on ice

-Spin at 1800 g, 4C, 5 min

-Remove s/natant and ressuspend in 4.25 ml CaCl2 (can stop for a bit here if necessary)

-Add 0.75 ml glycerol and mix gently with pippete (15% glycerol final)

-Flash freeze 50 ul aliquotes in a dry ice/EtOH bath

-Store at -80C