Competent cells (NEB stable)

Estimated time

Overnight + 3 hours + 2 days

Day 1. Start an overnight culture

Start a culture of the relevant bacterial strain (i.e., NEB Stable, ccdB resistant) from the glycerol stock stored in the same box as the aliquots of competent cells. Use ~4 mL plain LB media, and grow overnight at 30ºC.

Day 2. Prepare competent cells

Important

All pipetting should be done with filter tips, under a flame, and with fresh gloves to prevent contamination. Wipe down pipets with Obliterase prior to pipetting.

  1. Combine 1 mL of the overnight culture with 99 mL of fresh LB (plain) in a 500 mL Erlenmeyer flask.

  2. Shake at 37ºC until the OD (600 nm) measures 0.4-0.6 (0.5-0.6 is ideal). Use the Nanodrop to measure OD with default settings.

Note

Usually, it takes 2-2.5 hours to reach 0.4-0.6 OD (600 nm). Check the OD after ~90 min to estimate how much longer it will take—consider that bacteria double approximately every 30 min. Be careful not to overshoot! If so, you’ll need to restart the 100 mL culture.

  1. Once the OD (600 nm) reaches 0.4-0.6, place the cells on ice for at least 10 min.

  2. Using the Zymo Mix & Go! kit (stored in green bin in Deli fridge), prepare 10 mL of 1X Wash Buffer and 10 mL of 1X Competent Buffer by diluting the 2X solutions with Dilution Buffer. At this point, set the centrifuge to 4ºC to cool and label 100 Eppendorf tubes with the strain (e.g., “NS” for NEB Stable). Mark each labeled tube in the batch with a color different from those of any competent cells remaining in the freezer (the whole batch should be the same color).

  3. After incubating the culture on ice, pellet the cells by centrifugation at 3000xg for 10 min at 4ºC.

  4. Remove the supernatant completely and suspend the pellet GENTLY with 10 mL of ice-cold 1X Wash Buffer. Bleach the collected supernatant for 20 min and dispose down the sink.

  5. Re-pellet the cells by centrifugation at 3000xg for 10 min at 4ºC.

  6. Remove the 1X Wash buffer and collect it in a separate tube.

Important

To dispose of 1X Wash: Add a few mL of bleach to the collected 1X Wash Buffer. A dark brown/black precipitate will form. This can be left to settle over several days or can be centrifuged after 20 min of decontamination. Then, discard the supernatant down the drain and dispose of the tube with precipitate in the biowaste.

  1. GENTLY resuspend the cells in 10 mL of ice-cold 1X Competent Buffer.

  2. Aliquot the cell suspension ON ICE into the labeled Eppendorf tubes, 100 µL per tube.

  3. Store the cells in the relevant competent cell box at -80ºC (Elsa). Update the spreadsheet on the freezer door with the batch color and date.

  4. To confirm there is no plasmid contamination, innoculate LB + ampicilin and LB + kanamycin cultures with competent cells. It is convenient to use any leftover cells insufficient for a final aliquot for this step. Grow the cultures at 37ºC for two days.

Day 4. Check for cell growth

After two days in culture, check whether cells grew in the LB + ampicilin and LB + kanamycin conditions. If there is no growth, the competent cells are good to use. Mark the batch as such on the spreadsheet on the freezer door.

Important

If cell growth is observed, discard the entire batch of competent cells. This indicates likely plasmid contamination. Note the contamination on the spreadsheet on the freezer door and message the lab if any aliquots may have been used. To discard the competent cells, combine the contents of all the aliquots and follow the protocol for disposing of the 1X Wash Buffer waste.