Chemically Competent cells by the Rubidium Chloride method

Based on: https://wiki.epfl.ch/ptpsp/documents/Rubidium%20Chloride%20Competent%20Cell%20Protocol.pdf and https://wiki.epfl.ch/ptpsp/documents/competent_cells.pdf

Make the buffers fresh!

ADD RUBIDIUM AT THE END AS IT PRECIPITATES DURING pHING

TFB1

Dissolve potassium acetate and glycerol in H2O. pH to 5.8 with dilute (0.2%; 1M Acetic Acid). DO NOT OVERSHOOT. Dissolve metal salts. Filter 0.2 um sterile. Keep at 4C

TFB2

Dissolve MOPS and glycerol. pH to 6.5 with NaOH. DO NOT OVERSHOOT. Dissolve metal salts. Filter 0.2 um sterile. Keep at 4C

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Day 1

From a plate with correct selection markers, a preculture of cells were grown overnight at 37 with their appropriate antibiotics.

Day 2

Volume above of buffer are suitable for 500 mL of competent cells

1) Innoculate 500 mL of LB + antibiotics with 1:100 dilution of preculture. A good flask is 500 mL in 3L flask.

2) Grow until OD 0.4-0.6.

3) Transfer cells to 50 mL falcon tubes (prechilled). Let sit on ice 10 minutes. Spin down cells in 50 mL falcon tubes (prechilled) at 3900g x 15 minutes.

4) Decant supernatant

5) Resuspend each falcon tube with up to 20 mL of TFB1 (ie 200 mL for 500 mL of cells)

6) Let sit the resuspended cells on ice for 10-30 minutes.

7) Spin down cells at 3900g x 15 minutes.

8) Decant supernatant.

9) Resuspend ALL tubes with a total of 10-20 mL of TFB2 (ie 10-20 mL for 500 mL of cells; this makes 2-5% of initial culture volume)

10) Let sit the resuspended cells on ice for 30-60 minutes.

11) Aliquot 100 uL of cells per eppendorf. CHILL YOUR TUBES for best cells. Directly freeze in N2 then transfer to -80C.

Immediately test the cells and plate on antibiotic plates to make sure they have not picked up any resistance.

Immediately transform cells to determine their competency. Compare with cells from a previous batch

Previous times:

KL: 18.02.22 :  DH10EMBacY - 500 mL culture. 5 mL in 500mL of LB + Kan+Tet in 3L Flask. Started at 9:10. ready to be harvested 2H 45 minutes later. Resuspended in 10 mL, 100uL aliquots.