Making Competent Cells (using Zymo Mix and Go)

The following procedure is for a 50 ml E. coli culture in ZymoBroth™ (supplied with

T3001 only) or SOB medium; however, the volume can be adjusted according to your specific requirements. I often do larger volumes in the Vale lab, using the larger kit T3002.

1.    Day 1: Autoclave centrifuge bottles, or if you will be using fresh sterile bottles each time, please omit this step.

2.    Day 1 afternoon: Use a sterile 200 ul pipette tip to dip into a commercial competent cell tube, and streak it onto a plain LB agar plate. (Note this step is not strictly required but maybe good to get a single genetically pure isolate from the tube)

3.    Day 2 morning: Pick a single colony and inoculate 0.5 ml LB culture. Best to pick and grow 2-3 colonies in separate tubes to give yourself multiple overnight cultures at different dilutions.

4.    Day 2 evening: Inoculate the 0.5 fresh LB culture into 50mL ZymoBroth™ or SOB medium in a 500 ml culture flask. Best to inoculate 2-3 different cultures at different dilutions. Use 100, 250, and 500uL from starter tubes to do this. This allows you to get multiple chances at getting to the correct OD the next day in a reasonable time. Shake cultures vigorously (150 - 250 rpm) at 20 C until OD600nm is 0.4 - 0.6. Note that E. coli grows very slowly at this temperature. It usually take about 16 – 20 hours to reach the desired OD, depending on the stain.

5.    Day 3, Buffer Preparation Prior to Harvesting the Cells…The Wash and Competent Buffers are provided as 2X stock solutions. They need to be diluted to 1X by adding an equal amount of Dilution Buffer. To prepare 5 ml of 1X Wash Buffer: Add 2.5 ml Dilution Buffer and 2.5 ml of 2X Stock Wash Buffer. To prepare 5 ml of 1X Competent Buffer: Add 2.5 ml Dilution Buffer and 2.5 ml of 2X Stock Competent Buffer. Keep these freshly prepared 1X Buffers ice cold. It is important that each step of the following procedure should be done on ice or at 0-4°C.

6.   Pre-cool pipette tips in the cold room (Optional).

7.   Transfer the culture from Step 3 to ice. After 10 minutes, pellet the cells by centrifugation at 3,000 - 3,700 rpm (i.e., 1,600 - 2,500 x g) for 10 minutes at 0 – 4 C.

8.   Remove the supernatant and resuspend the cells gently in 5 ml ice-cold 1X Wash Buffer. Re-pellet the cells as in Step 2.

9.   Completely remove the supernatant and gently resuspend the cells in 5 ml ice-cold 1X Competent Buffer.

10.   Aliquot cell suspension into sterile PCR tubes using a multi-channel pipette in a cold room, or on ice.

11.   Snap freeze the cells liquid N2 and store below -70 C for transformation at a later time.