Competent cells (beth 1/29/02)

Prepare a day or two ahead of time: Fresh LB plate streak of cells, autoclave 200 eppendorfs, autoclave, 2 SS34 centrifuge tubes and 2 GSA tubes. Autoclave 1 liter flask with 100-200mLs LB. Make up 500mL Sterile filtered 0.1M MgCl2 and leave in cold room to keep cold at all times. Make up 500mL Sterile-filtered 0.1M CaCl2/14% Glycerol. Keep in cold room.

1) From a single colony or fresh streak of NON-competent cells, innoculate 5-10mls of LB O/N in a large test tube

2) the next day, split this 1:50 or 1:100 into 100 to 200mLs LB. Take a starting OD600

3) Grow until OD 600 is 0.34 to 0.4 DO NOT OVERGROW!

4) Chill on slushy ice for 10 minutes.

5) Spin at 4°C at 4000 rpm in the GSA for 10 minutes in sterile tubes. Keep the cells cold at all times.

6) Using sterile technique and keeping the cells on ice GENTLY resuspend in 1/2 the original volume in step 2 in sterile 0.1M MgCl2. Use a WIDE-Mouth pippette

7) Spin at 4°C at 4000 rpm in the GSA for 10 minutes in the same sterile tubes. Keep the cells cold at all times.

8) Using sterile technique and keeping the cells on ice GENTLY resuspend in 1/4 the original volume in step 2 in sterile 0.1M MgCl2. Use a WIDE-Mouth pippette

9) Chill on slushy ice for 25 minutes. Do not skimp on this time.

10) Spin at 4°C at 4000 rpm in the GSA for 10 minutes in the same sterile tubes. Keep the cells cold at all times.

11) Resuspend in 1/20^th the original volume in step 2 in sterile 0.1M CaCl2/14% glycerol. Use a WIDE-Mouth pippette and be GENTLE!

12) Aliquot into 50uL while keeping cold by either freezing in liquid nitrogen or dry ice/EtOH bath in the tube racks or aliquot into sterile tubes on ice and drop cells first into Liquid Nitrogen so that the cells do not freeze in the cap but in the bottom of the tube. Once the liquid nitrogen vat gets full, transfer done tubes to a box and place in –80°C until you have aliquoted all of the cells. Place in the appropriate rack in the freezer with a note about what color tubes are what.