Team:UNICAMP-Brazil/Protocols/Preparation of electrocompetent E. coli

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Preparation of electrocompetent E. coli

1. Preferably, select single colony of E. coli from fresh LB plate to inoculate a 100 ml LB overnight (O/N) starter culture. Grow the starter culture O/N in 37°C shaker (250rpm).

2. Inoculate 1L of LB media and place culture in 37° shaker. Grow cells and measure OD600 every 30min. When the OD600 equals 0.5-0.8 (log phase growth), remove the cells from the shaker and place on ice.

NOTE: It very important to keep the cells at 4°C (or on ice) for the remainder of the procedure.

3. Split the 1L culture into four equal parts by pouring ~250ml of culture into each chilled 250ml Corning pointed bottle.

4. Spin in GPR centrifuge at 4000rpm, 15min at 4°C.

5. Place bottles on ice. Remove supernate immediately as cell pellet begins to lift off quickly. Gently resuspend each pellet in 250ml ice-cold 10% glycerol.

6. Spin in GPR centrifuge at 4000rpm, 15min at 4°C.

7. Place bottles on ice. Remove supernate. Gently resuspend each pellet in 250 ml of ice-cold 10% glycerol.

8. Spin in GPR centrifuge at 4000rpm, 15min at 4°C.

9. Place bottles on ice. Remove supernate. Gently resuspend each pellet in 10ml ice-cold 10% glycerol.

10. Spin in GPR centrifuge at 4000rpm, 15min at 4°C.

11. Place tubes on ice. Remove supernate. Gently resuspend each cell pellet in 1ml of ice-cold 10% glycerol.

12. With cell suspensions on ice, prepare aliquots of 40 ul of cells in pre-chilled 1.5ml eppendorf tubes. Snap freeze tubes containing cells in liquid N2. Store frozen cells at -80°C.