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Prepare chemically competent E. coli cells

Goal

Make a lab stock of chemically competent E. coli cells for routine plasmid cloning. We typically use NEB Stable cells (ecHP56) for cloning in our lab.

Method I. Zymo Research Mix & Go! Kit

This protocol adapts the Mix & Go! kit from Zymo Research.

Materials

  • Mix & Go! E. coli transformation kit (Zymo Research cat. no. T3001)
  • Frozen stock of NEB Stable cells (ecHP56)
  • LB (liquid and plates)
  • SOB (liquid, 100 mL, recipe below)

Procedure

  1. (Day 1) Streak ecHP56 on an LB plate and incubate at 37 °C overnight.
  2. (Day 2) Inoculate a single colony of ecHP56 in liquid LB and incubate overnight at 37 °C with shaking or rotation.
  3. (Day 3) The next morning, in a baffled flask, inoculate 100 mL of SOB with 1 mL of the overnight culture. Shake at 20 °C, 180 rpm, in a temperature-controlled Innova shaker (Strong lab).
  4. Measure the OD600 after 4 hours, and every hour thereafter, until cells reach OD600 between 0.3 and 0.5. Also turn on a clinical centrifuge and chill to 4 °C.
  5. When cells reach target OD, immediately place the flask on ice for 10 minutes, and chill two 50-mL conical tubes on ice.
  6. While cells and tubes are chilling, prepare 5 mL of each Mix & Go! buffer solution in separate 15-mL conical tubes, by diluting 2.5 mL of 2x wash buffer, and 2.5 mL of 2x competent buffer, with 2.5 mL of dilution buffer. Store these solutions on ice.
  7. In a separate bucket, prepare a rack of ~100 PCR tubes (at least 12 x 8-tube strips) in an ice-water bath.
  8. After cells have chilled for 10 minutes on ice, split culture between chilled 50-mL conical tubes and pellet 10 minutes at ~3,000 g (max clinical centrifuge speed).
  9. Remove supernatant and place 2.5 mL of ice-cold wash buffer on each pellet. Resuspend pellets with a 1-mL pipet tip.
  10. Transfer washed cells to a cold 15-mL conical (you can reuse the wash buffer tube for this) and pellet again for 10 minutes at ~3,000 g.
  11. Completely and carefully remove the supernatant—the pellet will be somewhat loose—and gently resuspend in 5 mL of competent buffer, using a 1-mL pipet tip.
  12. Dispense ~100 50 μL aliquots of resuspended cells into chilled PCR tube strips on ice, and place immediately in a box at -80 °C.

SOB recipe

component amount (g)
Yeast extract 5
Tryptone 20
NaCl 0.584
KCl 0.186
MgSO4 2.4
  1. Add reagents to 1L tap water.
  2. Adjust to pH 7 with concentrated NaOH.
  3. Dispense into 100 mL aliquots and autoclave 45 min on a liquid cycle.

Method II. Homemade Transformation Buffer

Materials

  • Transformation Buffer
component amount alt reagent
HEPES 236mg
KCl 186mg
CaCl2.2H2O 290mg 219mg CaCl2
ddH2O to 100ml
pH to 6.7 with KOH (need about 6-10ul of 10M KOH)
MnCl2.H2O 840mg 1156mg MnCl2.4H2O
  • Filter through 0.2µ filter.

  • Make fresh and store at 4C.

  • SOB Medium

(See recipe above)

Procedure

  1. Day 0: Streak NEB-Stable cells (ecAS4.1 or ecHP56) on LB plates and incubate at 37°C, 16-19 hours.
  2. Day 1: Start overnights of a single colony of the strain, LB, 37°C, 5ml, 200rpm, 12–17 hours.
  3. Day 2: Dilute overnights (1:5 to 1:20) into 5mL LB and shake in 23-25C waterbath, 200rpm, 5-6hrs (want these to be in approx log phase in the afternoon)
  4. Day 2: After 5-6hrs measure the OD600 and dilute into 1L SOB accordingly for 23C 200rpm Strong lab shaker in 2.8L baffled flask (need to sign up a few days beforehand).
    1. General benchmark: ecHP56 cells double once every ~1.32 hours at 23C, so if you want to harvest in 19-20hrs, then dilute into 1L SOB for a final OD600 of 0.00002 (~14.5doublings)
    2. Example: if you measured the culture's OD600 to be 1.0, then do: (vol)(OD600 1.0) = (1L)(0.00002) -> vol = 20uL of culture into 1L SOB at 23C 200rpm should reach OD 0.4-0.5 in 19-20hrs
    3. Note that the spectrometer's most reliable readings are between 0.1 and 1.0, so you will likely need to make dilutions prior to measuring
  5. Day 3: Check OD600 every half hour ~2hrs before you think they should be ready (e.g., if you diluted for 19-20hrs, check at 17hrs)
    1. Bring the cuvette/pipette to the incubator and take an aliquot for measurement quickly
  6. While waiting for OD600 to reach 0.4-0.5, prechill the following reagents, place in cold-room:
    1. 4 × 500ml bottles on ice
    2. transformation buffer on ice
    3. 2 x 50mL conicals on ice
    4. PCR strip tubes
    5. Set beckman centrifuge to 4C to prechill (include the yellow 50mL conical holders so they can prechill too)
    6. Prepare an ice+water bath large enough to fit the 1L flask of cells
  7. When culture hits OD600\~0.4—0.5 rapidly chill your cultures by placing them in the ice-water bath in cold-room for 10 min and swirling them occasionally.
  8. Aliquot the cells into the 4×500ml pre-chilled bottles (equilibrate the weights by eye, \~250mL each) and spin 3250g, 20min, 4°C. Pour off supernatant.
  9. Resuspend the pellet in 10ml ice-cold TB by pipetting, transfer to the 2 x 50mL conicals (20mL/conical). Incubate in ice-water bath for another 10 min. Make sure that the centrifuge is cold.
  10. Spin down cells 3250g, 20min, 4°C. Pour off supernatant.
  11. Resuspend cells in 1-5ml of ice-cold TB and combine them.
  12. Bring up the total volume to 16.6mL with TB, then add 1.2ml of molecular biology grade DMSO.
    1. Aliquot the DMSO at room-temp, then bring to cold-room just before adding or it will solidify
  13. Mix well gently by pipetting and leave on ice for 10 min.
  14. Aliquot 50uL/100μl into pre-chilled PCR strip tubes with individual caps and place immediately in −80°C box.

Checking transformation efficiency of competent cells

Allow cells to freeze overnight at -80 °C before testing competence.

Method I. Use NEB pUC19 to test Mix & Go! cells

  1. Thaw 2 x 50 μL aliquot of cells on ice.
  2. Mix 1 μL of NEB pUC19 transformation control (50 pg/μL) with one 50 μL aliquot of competent cells.
  3. Mix 1 μL of TE with the other 50 μL aliquot of competent cells.
  4. Incubate on ice for 5 minutes.
  5. Plate directly onto prewarmed LB + carbenicillin plates (you can use one plate, divided in half, for both transformations), and incubate overnight at 37 °C.
  6. Verify there are no colonies in the no-plasmid control. Then count colonies (CFUs) and note the transformation efficiency. Calculate efficiency in transformants per μg as (number of CFUs) x (100,000).

Method II. Older method with homemade competent cells

  1. Mix 100ng of MG1655+pUC19 with 50µl of competent cells.
  2. Incubate on ice, 30 min.
  3. Heat shock 42°C, 45s.
  4. Incubate ice, 2 min.
  5. Recover in 500ul LB, 1 hour, 37°C, 200rpm in culture tubes.
  6. Dilute 10µl of recovered cells into 1ml of LB -> 1:100
  7. Dilute 10µl of above diluted cells again into 1ml of LB.
  8. Dilute 10µl of above diluted cells again into 1ml of LB.
  9. Plate 100µl of the three dilutions on pre-warmed carb100 plates and incubate overnight at 37°C.

  10. Count colonies and note the transformation efficiency below with the date as CFU / 1µg of pUC19 / 50µl of competent cells.

  11. Dilution used = 10uL transformants / 500 recovered vol * 100uL plated / 1000uL diluent = 0.002

Transformation Efficiencies (note with each prep)

Prep by Prep date Method pg pUC19 plated CFUs Efficiency (CFUs/µg pUC19) -80° C Box
kchen July 2020 Homemade ? 6 30,000 chemically competent e.coli
kchen Sept 2020 Homemade ? 190 950,000 Chem. competent e.coli Box 2
ton/kchen Feb 2022 Homemade ? 10,000 100,000 Chem. competent e.coli Box 2
jyelland Oct 2024 Zymo Kit 10 ~100 ~10,000,000 Chem. competent e.coli Box 2