Competent cells (NEB stable)

Day 1. Culture NEB stable cells for overnight.

Take the glycerol stock of NEB stable from -80C freezer (Elsa) and pick the cells and culture them with 2ml of plain LB at 30C for overnight.

Day 2. Grow the competent cells and stock the cells.

  1. Take the 0.5ml of the overnight NEB stable cells from Day 1, and put the cells into 50ml of plain LB in a 250 mL Erlenmeyer flask and culture them @ 30C until the OD(600nm) reaches 0.4-0.6 (use nanodrop to measure the OD).

Note

Usually, it takes 2-2.5 hours to the OD(600nm) reaches 0.4-0.6 when the cells are cultured at 30C.

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

  2. Prepare 5ml of 1xWash Buffer and 5ml of 1xCompetent Buffer by diluting the 2x solutions with Dilution Buffer.

  3. From step 2, after 10 minutes incubation on ice, pellet the cells by centrifugation at 3000xg for 10min. at 4C.

  4. After the cenritugation, remove the supernatant completely and suspend the pellet GENTLY with 5ml of ice-cold 1xWash Buffer prepared at step 2.

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

  6. After removing the 1xWash Buffer COMPLETELY by transfering the supernatant to an another 50ml falcon tube (see below how to descard the wash buffer), GENTLY suspend the cells with 5ml of ice-cold 1xCompetent Buffer.

Important

Add a few ml of 10% bleach to the collected 1xWash Buffer and centrifuge the tubes at 3000xg for 3-5min.. Then, you can discard the supernatant into a sink and the precipitate to a biowaste box.

  1. Aliquot 100ul of the cell suspention (ON ICE!) into eppendorf tubes. Mark each labeled tube with Sharpie that has a unique color from any competent cells remaining in the freezer.

  2. Store the cells @ -80C (ELSA)

  3. To make sure if the competent cells are not carrying any plasmid, culture the competent cells made at step 8. with 2ml of LB-Kan, LB-Amp, and, LB-Chlor for about 2days at 30C.

Day 4. Confirm the cell growth

  1. Confirm if the competent cells were not growing in any of the media. If you see cell growth in either LB media, discard the competent cells you made and remake the cell from step 1.