Team:UNICAMP-EMSE Brazil/protocols/chimiocompetent prepare

Preparation of Chemically competent E. coli cells

A) Lab materials and solutions

1) TYM (for 1 L)

Tryptone	20 g
Yeast extract	5 g
NaCl	5.8 g
MgSO4.7H2O	2.5 g

• AUTOCLAVE

Prepare more than 1 Erlenmeyer for each volume (for security reasons)

2) TFB1 and TFB2 (storage under refrigeration)

TFB1		for 1L	for 200 mL
KOAC (Potassium acetate)	0.03M	2.94 g	0.59 g
MnCl2.4H2O	0.05M	9.90 g	1.98 g
KCl	0.10M	7.46 g	1.49 g
CaCl2.2H2O	0.01M	1.47 g	0.29 g
Glycerol (glycerin)	15%(m/V)	150 mL	30 mL

• 100 ml of this solution will be used in the procedure
• Filtration required (0.45µm filters) – In the flux! (for sterilization)

TFB2		for 1L	for 200 mL
MOPS	0.01M	2.04 g	0.41 g
KCl	0.01M	0.75 g	0.15 g
CaCl2.2H2O	0.075M	11.03 g	2.21 g
Glycerol (glycerin)	15%(m/V)	150 mL	30 mL

• Adjust the pH to 7 with KOH
• Filtration required (0.45µm filters) – In the flux! (for sterilization)

3) 1 culture plate with solid LB medium WITHOUT ANTIBIOTIC !

To grow the colonies from the stock of competent cells

B) Material to be checked or prepared in advance

1- Prepare the solutions / Place Eppendorf and Falcon in the freezer

2- Make reservation for the following equipments:

- Incubator 37°C (for colonies growth in the culture plate)

- Shaker 37°C (for cells growth)

- Spectrophotometer (measures at OD600)

- Table centrifuge (for 50 mL Falcon)

- Laminar Flux hood

C) Proceedings

1) 1st day: Inoculate cells in the with solid LB medium without antibiotic (37°C) –overnight (inoculate by the end of the afternoon)

2) 2nd day: Select colonies and let them grow overnight in a 40 mL Erlenmyer with TYM (shaker 37°C, 200 rpm)

3) 3rd day:

- Inoculate 100 µL from the cells culture (grown overnight) in 10 mL of TYM

- Grow the 10 mL culture until it reach the OD600=0.2-0.6 (60 to 120 min.)

- Add the entire content of the Erlenmyer (10mL) into another Erlenmyer containing 40 of TYM

- Grow the culture until it reach the OD600=0.5-0.9 (60 to 120 min.)

- Add the entire content of the Erlenmyer (50mL) into another Erlenmyer containing 200 of TYM

- Grow the culture until it reach the OD600=0.6 (90 to 150 min.)

- Grow the culture until it reach the OD600=0.6 (90 to 150 min.)

- Cool down quickly (ice bath) until it reach the desired OD!

1. Centrifuge the cells at 4000 rpm (in 50 mL Falcon tubes) during 15 minutes at 4°C.

2. Discard the supernatant, resuspend the cell pellet GENTLY in 100mL ice cold TFB1 (initiate with 2mL for each tube, pass through only 2 tubes and complete each tube for 50mL )

3. Centrifuge for 8 minutes at 4°C (4000 rpm)

4. Resuspend GENTLY in 10mL ice cold TFB2 (initiate with 2mL for each tube, put it all together in one tube only and complete the volume to 10 mL with TFB2)

5. Aliquot 100µl/Eppendorf tube (COLD, new and sterile)

 Flash freeze in liquid nitrogen and store in the -80°C freezer