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A Quick and Easy Method for Making Competent Escherichia coli Cells for Transformation Using Rubidium Chloride   

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Abstract

This protocol describes a quick and efficient method to make competent E. coli cells for transformation using rubidium chloride. Commercial competent cells are expensive and this protocol provides a cheaper alternative to them.

Keywords: Competent cells, E. coli, Transformation efficiency, TOP10, DH5α

Background

The success of gene cloning is highly dependent on the transformation efficiency of bacterial cells. The efficiency can be artificially improved by treating the cells with chemicals or electric pulses. Several protocols are available to prepare competent E. coli cells, however, they are usually long, laborious, and show inconsistency in competence. The protocol by Green and Rogers (2013) overcomes these downsides and allows the preparation of highly competent cells (~106-108 CFU/µg DNA). While other protocols require cells to be grown at low temperature (19-22 °C), this protocol involves growing cells at 37 °C. Thus, the cells grow faster and reach log phase within 4 h as compared to 18-24 h. This protocol is highly reproducible.

Materials and Reagents

  1. 25 mm, 0.2 µm syringe filter PES (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 725-2520 )
  2. 5 ml syringe (Fisher Scientific, catalog number: 14-829-45)
    Manufacturer: BD, catalog number: 309646 .
  3. 1.5 ml Eppendorf tubes (Corning, Costar®, catalog number: 3207 )
  4. 100 mm Petri dish (Corning, Falcon®, catalog number: 351029 )
  5. Pipettes tips
  6. 10 ml culture tubes (VWR, catalog number: 60818-725 )
  7. Spreaders (Fisher Scientific, catalog number: 14-665-230 )
  8. 250 ml centrifuge bottles (Sigma-Aldrich, catalog number: Z353736)
    Manufacturer: Thermo Fisher Scientific, catalog number: 3141-0250 .
  9. E. coli strain (TOP10 or DH5α)
  10. Control plasmid (e.g., pUC19)
  11. LB broth (Fisher Scientific, catalog number: BP9723-500 )
  12. LB agar (BD, catalog number: 244510 )
  13. Ice
  14. Liquid nitrogen
  15. Rubidium chloride (RbCl) (Sigma-Aldrich, catalog number: R2252 )
  16. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: 203734 )
  17. Potassium acetate (Sigma-Aldrich, catalog number: P1190 )
  18. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C8106 )
  19. Glycerol (Fisher Scientific, catalog number: BP229-4 )
  20. MOPS (Fisher Scientific, catalog number: BP308-100 )
  21. Acetic acid (Fisher Scientific, catalog number: A38-212 )
  22. Sodium hydroxide (Fisher Scientific, catalog number: S318-1 )
  23. Vector specific Antibiotics
  24. Buffer I (see Recipes)
  25. Buffer II (see Recipes)

Equipment

  1. Pipettes (P1000 and P200)
  2. 1 L flasks (Fisher Scientific, catalog number: 10-040K)
    Manufacturer: Corning, catalog number: C49801L .
  3. Spectrophotometer (Beckman Coulter, model: DU-640 , catalog number: 8043-30-1090)
  4. Water bath (Marshall Scientific, model: Precision 181 )
  5. 37 °C incubator and shaker (Thermo Fisher Scientific, model: MaxQTM 4000 , catalog number: SHKE4000-1CE)
  6. Centrifuge (Thermo Fisher Scientific, SorvallTM, model: RC-5B , catalog number: 8327-30-1004)
  7. pH meter (Fisher Scientific, catalog number: 13-644-928)
    Manufacturer: Thermo Fisher Scientific, catalog number: 1112106 .

Procedure

  1. Prepare competent cells
    Note: All steps are carried out in sterile conditions.
    1. Day 1 (1 h preparation + overnight)
      Streak E. coli strain (e.g., TOP10 or DH5α) on an LB agar plate in a 3 streak fashion (Reference 2) and incubate overnight at 37 °C.
    2. Day 2 (10 min preparation + overnight)
      Pick a single E. coli colony and culture in 5 ml LB broth in a 10 ml culture tube at 37 °C overnight at 200 RPM.
    3. Day 3 (7 h)
      1. Inoculate 500 ml LB broth in a 1 L flask with 500 µl overnight E. coli culture and grow at 37 °C (200 RPM) until OD600 = 0.5 (about 4 h).
      2. Chill cells on ice for 10 min.
      3. Centrifuge E. coli cells at 2,000 x g for 10 min at 4 °C.
      4. Remove supernatant and resuspend the pellet in 50 ml ice-cold buffer I (see Recipes). Keep the cells on ice for the entire time and shake manually in circular motions on ice. It will take 20-30 min to resuspend the cells (Video 1).
        Note: Do not pipette the cells up and down, but you may break the pellet with a pipette for faster resuspension.
      5. Centrifuge cells at 2,000 x g for 10 min at 4 °C.
      6. Remove supernatant and resuspend the pellet in 25 ml ice-cold buffer II (see Recipes) by shaking. Keep the cells on ice for the entire time. It will take some time (20-30 min) to resuspend the cells (Video 1).
        Note: Do not pipette the cells up and down, but you may break the pellet with a pipette for faster resuspension.

        Video 1. Resuspending pellet in buffer I or buffer II: Gently rotate bottles in an ice-bucket to resuspend the bacterial pellet in buffer

      7. Aliquot 50 μl cells into pre-chilled Eppendorf tubes on ice and immediately freeze the tubes in liquid nitrogen. Work quickly through this step.
      8. Store competent cells at -80 °C.

  2. Check competence of cells
    1. Day 1 (2 h preparation +overnight)
      1. Thaw two tubes of competent cells on ice for 5 min.
      2. Add 1 ng positive control plasmid (pUC19) to one tube and 1 μl water to the other tube as a negative control.
      3. Incubate on ice for 30 min.
      4. Heat-shock the cells at 42 °C in a water bath for 30 sec and immediately put the tubes on ice for 2 min.
      5. Add 200 μl LB broth to the cells and grow the cells at 37 °C for one hour at 200 RPM.
        Note: Fresh LB is not necessary at this step, however you must use uncontaminated LB.
      6. Plate 50 μl (1/5th of total volume) on an LB agar plate with appropriate antibiotic.
      7. Grow the cells overnight at 37 °C.
    2. Day 2 (30 min)
      Check the efficiency of competent cells by counting the number of colonies on both positive and negative control.
      Note: If the transformation efficiency is high and there is a high number of evenly spread colonies, you may count colonies on a fourth of the plate area and multiply the value by 4 for an approximate total number of colonies. Else, you may start with a lower concentration of control plasmid (1 pg-1 ng). The negative control should have no colonies as any colonies found indicate contamination with an antibiotic-resistant strain.

Notes

This protocol is highly reproducible if you:

  1. Keep the cells on ice at all times and work fast through the process.
  2. Prepare buffers fresh on the day of the competent cells preparation.
  3. Work with small culture volumes (e.g., 200 ml) to shorten the duration of the preparation.

Recipes

  1. Buffer I (prepare 50 ml per 500 ml culture)
    Final concentrations:
    100 mM RbCl (12 g/L)
    50 mM MnCl2·4H2O (9.89 g/L)
    30 mM potassium acetate (2.94 g/L)
    10 mM CaCl2·2H2O (1.47 g/L)
    15% (v/v) glycerol (150 ml/L)
    Adjust pH to 5.8 with dilute acetic acid and filter sterilize
    Note: Store at 4 °C, protect from light.
  2. Buffer II (prepare 25 ml per 500 ml culture)
    Final concentrations:
    10 mM MOPS (2.09 g/L)
    10 mM RbCl (1.2 g/L)
    75 mM CaCl2·2H2O (11 g/L)
    15% (v/v) glycerol (150 ml/L)
    Adjust pH to 6.5 with dilute NaOH and filter sterilize
    Note: Store at 4 °C and protect from light.

Acknowledgments

This protocol is adapted from Green and Rogers (2013). I would like to thank Bergmann lab members for fine-tuning this protocol.
The authors declare that there are no conflicts of interest.

References

  1. Green, R. and Rogers, E. J. (2013). Chemical transformation of E. coli. Methods Enzymol 529: 329-336.
  2. Streaking and Isolating Bacteria on an LB Agar Plate (https://www.addgene.org/protocols/streak-plate/)
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Sharma, N., Anleu Gil, M. X. and Wengier, D. (2017). A Quick and Easy Method for Making Competent Escherichia coli Cells for Transformation Using Rubidium Chloride. Bio-protocol Bio101: e2590. DOI: 10.21769/BioProtoc.2590.
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