Team:UANL Mty-Mexico/Results/Protocolstupapaingles

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Protocols

  • Preparation of E. coli Calcium Chloride competent cells
      1. Inoculate a single colony into 5 mL of LB media without any antibiotics and grow overnight at 37 °C with vigorous shaking.
      2. Inoculate 1 mL of the desired strain into 100 mL of fresh LB, use a 500 mL flask.
      3. Incubate at 37 °C with vigorous shaking until 0.3 - 0.4 sideOD600
      4. Put the flask on ice. Pre-chill 50 mL centrifuge tubes and the centrifuge itself at 4°C.
      5. Centrifuge 50 mL of the culture at 8,000 rpm for 5 minutes at 4 °C.
      6. Remove the supernatant and add 10 mL of cold CaCl2 0.1 M. Vortex until the pellet is resuspended.
      7. Incubate on ice for 30 minutes, shake the tube once in a while.
      8. Centrifuge at 8,000 rpm for 5 minutes at 4°C. Remove the supernatant and add 2 mL of CaCl2 0.1 M. Resuspend carefully using a micropipette. Keep always on ice.
      9. Mix the two preparations in a tube and store on ice, or use for transformation.

      Note: The competent cells can be stored on ice up to two weeks.


  • Preparation of E. coli Rubidium Chloride competent cells
      1. Inoculate 5 mL of LB broth with DH5α and incubate the culture overnight at 37°C with vigorous shacking.
      2. Use small culture to inoculate 100 mL of LB broth. Incubate at 37°C with shacking until the culture reaches an optical density (OD595) 0.4-0.6.
      3. Transfer to two 50 mL centrifuge tubes.
      4. Spin at maximum speed for 5 minutes at 4°C.
      5. Remove supernatant.
      6. Add 20 mL of TBF1 and resuspend the pellet.
      7. Incubate on ice for 20 minutes.
      8. Pour off supernatant.
      9. Centrifuge 5 minutes at 8000 rpm/4°C.
      10. Resuspend cellular pellet with 4 mL of TBF2.
      11. Place aliquots of 100 µL at -70°C.
  • Heat-shock transformation of E. coli competent cells
      1. Add 50 µL of Ca+2 competent cells to a pre-chilled centrifuge tube. Keep always on ice until step 4.
      2. Add plasmid DNA (100 ng) or ligation (up to 5 µL) depending on DNA concentration.
      3. Use 1 µL of a 1 ng/µL DNA sample as positive test in a separate tube. It is recommended to use a DNA-free negative test tube as well.
      4. Chill the tube on ice for 20 - 30 minutes.
      5. Expose the reaction mixture to a 42ºC 1 minute heat-shock.
      6. Put the tube on ice for 2 minutes.
      7. Add 200 µL of antibiotic-free LB media.
      8. Incubate at 37ºC for 20 - 30 minutes.
      9. Spread the appropriate quantity of cells (50-200 µL) on selective LB agar plates.
      10. Incubate overnight at 37º C.
      11. The positive plate must have around 1,000 colonies as an optimal (1X106 transformants per µg supercoiled DNA).

      Notes: Until heat-shock, handle the tubes from the upper part to avoid warming the cells. Low temperature is critical for successful transformation. Avoid transforming with more than 5 µL of ligation mixture, as ligation buffer may reduce transformation efficiency.


  • Preparation of Electrocompetent E. coli cells
      1. Inoculate a single colony of E. coli in 5 mL of LB media. Grow overnight or for 5 hours at 37°C with shaking at 250 rpm.
      2. Inoculate 2.5 mL of the previous culture in 200 mL of LB media in a 2 L flask. Grow at 37 °C shaking at 300 rpm until the culture reaches an OD of 0.5 - 0.7.
      3. Chill the cells on ice for 10 - 15 minutes and then transfer the cells into a pre-chilled centrifuge bottle.
      4. Centrifuge at 4,200 rpm for 10 minutes at 2 °C (Beckman J-6M).
      5. Remove the supernatant and resuspend the pellet in 5 mL of cold water. Add 200 mL of cold water and mix well. Centrifuge at 4,200 rpm for 10 minutes at 2 °C.

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