Team:Cambridge/Protocols/Making Competent Cells

Making Electro-Competent Bacterial Cells

Competent cells are those which will take up extra-cellular DNA and express it. This technique is central to synthetic biology.

Theory

While none of the mechanisms by which cells take up new DNA have been well characterised, the conditions required in order for the mechanism to take place are well documented. This generally requires placing the cells under specific stresses which causes this to take place. Once prepared, competent cells can be stored indefinitely at -80 degrees celsius.

Preparing E. coli for transformation by electroporation

The aim of the procedure is to remove salts that might interfere with electroporation. Cells are grown without selection so it is very important to keep sterility, especially in the early stages. Once the cells have been pelleted it is important to do everything on ice (chill solutions and centrifuge before use), and avoid vortexing or otherwise abusing the cells. Use pipette tips with the ends removed (make sure these are sterile!); forcing cells through narrow apertures can induce lysis.

Materials and Equipment

To make 30x 100μl eppendorfs of competent cells in 10% glycerol stock:

• 250ml LB
• 200ml hepes
• 105ml 10% glycerol
• p1000 pipette tips with ends removed, sterilized (alternatively, ends can be removed on demand using scissors sterilized with ethanol and a flame)
• a chilled centrifuge (at 4C).

Autoclave all solutions and glassware before use. Chill the hepes and glycerol to 4°C and set the temperature on the centrifuge to 4°C before you perform the washes.

Method

• Grow an overnight culture of cells in LB. The next day make a 1 in 1000 dilution into fresh medium. Each 100ml of this new culture will make about 1ml of glycerol stock of competent cells (10x 100%mu;l eppendorfs).
• Grow this culture to an OD600 of 0.5, using plain LB to calibrate the spectrophotometer. Discard the aliquots used for taking the readings.
• From now on perform all steps on ice or at 4°C.
• When the culture has an OD600 above 0.5, decant the culture into 50ml falcon tubes. Pellet the cells by centrifugation at 3000rpm for 15 minutes at 4°C.
• Discard the supernatant and resuspend the pellet gently in the same volume of (chilled) hepes. Pellet the cells by centrifugation at 3000rpm for 10 minutes at 4°C.
• Discard the supernatant and resuspend the pellet gently in half the volume of (chilled) hepes. Combine the cells into half the number of falcon tubes if desired. Pellet the cells by centrifugation at 3000rpm for 10 minutes at 4°C.
• Discard the supernatant and resuspend the pellet gently in the same volume of (chilled) 10% glycerol as the volume used in the previous step. Pellet the cells by centrifugation at 3000rpm for 10 minutes at 4°C.
• Gently resuspend each pellet in ~1.5ml of 10% glycerol.
• Using the sawn-off pipette tips, pipette 100μl of the resuspended cells into as many eppendorf tubes as possible. Place these in a rack in a bath of ice water to keep the cells cold.
• Transfer the rack to -80°C for storage. The cells are viable for months or a few years.

In other bacteria

The conditions required for other bacteria to be competent vary. Some bacteria are naturally competent.

Safety

All equipment (including gloves) that may have come into contact with the bacteria must be autoclaved.

Back to Protocols