Team:UNICAMP-EMSE Brazil/protocols/thermocompetent prepare

Preparation of Thermocompetent E. coli

Making competent cells:

• MATERIALS

• Buffers and Solutions
• CaCl2•2H2O (1 M)
• MgCl₂ - CaCl₂ solution, ice cold
• Media
• SOB medium for initial growth of culture
• SOB agar plates containing 20 mM MgSO₄ and the appropriate antibiotic
• SOC medium

• RECIPES

• NaCl

• To prepare a 5 M solution: Dissolve 292 g of NaCl in 800 ml of H2O. Adjust the volume to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl solution at room temperature.

• SOB

• deionized H2O, to 950 ml
• tryptone, 20 g
• yeast extract, 5 g
• NaCl, 0.5 g

• Shake until the solutes have dissolved. Add 10 ml of a 250 mM solution of KCl. (This solution is made by dissolving 1.86 g of KCl in 100 ml of deionized H2O.) Adjust the pH of the medium to 7.0 with 5 N NaOH (approx. 0.2 ml). Adjust the volume of the solution to 1 liter with deionized H2O. Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle. Just before use, add 5 ml of a sterile solution of 2 M MgCl2. (This solution is made by dissolving 19 g of MgCl2 in 90 ml of deionized H2O. Adjust the volume of the solution to 100 ml with deionized H2O and sterilize by autoclaving for 20 minutes at 15 psi [1.05 kg/cm2] on liquid cycle.)

• SOC

• deionized H2O, to 950 ml
• tryptone, 20 g
• yeast extract, 5 g
• NaCl, 0.5 g

• For solid medium, please see Media Containing Agar or Agarose. SOC medium is identical to SOB medium, except that it contains 20 mM glucose. After the SOB medium has been autoclaved, allow it to cool to 60°C or less. Add 20 ml of a sterile 1 M solution of glucose. (This solution is made by dissolving 18 g of glucose in 90 ml of deionized H2O. After the sugar has dissolved, adjust the volume of the solution to 100 ml with deionized H2O and sterilize by passing it through a 0.22-μm filter.)

• METHOD

1. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 hours at 37°C. Transfer the colony into 100 ml SOB medium (LB-may be used) in a 1-liter flask. Incubate the culture for 3 hours at 37°C with vigorous agitation, monitoring the growth of the culture. As a guideline, 1 OD600 of a culture of E. coli strain DH1 contains approx. 109 bacteria/ml.
2. Transfer the bacterial cells to sterile, disposable, ice-cold 50-ml polypropylene tubes. Cool the cultures to 0°C by storing the tubes on ice for 10 minutes.
3. Recover the cells by centrifugation at 2700g for 10 minutes at 4°C.
4. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away.
5. Resuspend each pellet by swirling or gentle vortexing in 30 ml of ice-cold MgCl2-CaCl2 solution.
6. Recover the cells by centrifugation at 2700g for 10 minutes at 4°C.
7. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away.
8. Resuspend the pellet by swirling or gentle vortexing in 2 ml of ice-cold 0.1 M CaCl2 for each 50 ml of original culture.

When preparing competent cells, thaw a 10-ml aliquot of the CaCl2 stock solution and dilute it to 100 ml with 90 ml of pure H2O. Sterilize the solution by filtration through a prerinsed Nalgene filter (0.45-μm pore size), and then chill it to 0°C.

1. At this point, either use the cells directly for transformation or make aliquots of 1 mL and mixed with 30% glycerol in pre-chilled (in liquid nitrogen) eppendorf tube and store at -70 °C.