Transformation is a common method in molecular biology with many applications, including cloning, DNA sequencing, and DNA library construction. To perform transformation, you must use competent cells. This article provides information about how to use competent cells, types of competent cells, the common steps to make competent cells, and competent cell storage.
In This Article:
3 Basic Steps of Transformation
There are three basic steps in many protocols to transform bacterial cells (Aune & Aachmann, 2010):
1. The preparation step: the bacterial cells are made competent to uptake foreign DNA by modifying the permeability of the cell membrane and the cell wall.
2. The transformation step: the transformation step is performed to allow DNA (usually plasmid DNA) to enter the cell. The most common transformation methods are electroporation or heat shock transformation.
3. The recovery step: the cells are incubated in a recovery medium to restore the cell membrane and the cell wall.
Electroporation vs. Heat Shock Transformation
The advantages of using electroporation are the higher efficiency, more colonies, and much faster transformations compared to heat shock method.
Transformation efficiencies for electroporation are 5.0 x 109 – 2.0 x 1010 CFU/µg DNA, whereas the efficiencies for heat shock transformation are 1.0 x 105 – 2.0 x 109 CFU/µg DNA (Aune & Aachmann, 2010). Therefore, electroporation is helpful when you have to construct DNA libraries.
The drawback of this method is that you must have an electroporator, which is a special piece of equipment. In addition, the common problem during electroporation is the presence of salts or air bubbles in your DNA, and in the cuvette, can cause an arcing. Unfortunately, this will make you lose your sample and require you to redo your ligation reaction.
To learn more about how to prevent an arcing, find GoldBio article:
Heat shock transformation is relatively easy compared to electroporation. It is also simple, only requiring a water bath. You can use this method, when you only need to get a few positive clones.
To perform transformation, you must have competent cells. There are two types of artificially competent cells available: electrocompetent and chemically competent. What you use for electroporation is electrocompetent cells, whereas chemically competent cells are used for the heat-shock transformation method.
What are Electrocompetent Cells and Chemically Competent Cells?
Competent cells are bacterial cells commonly used for transformation. Transformation of bacteria involves the binding of foreign DNA to the cell membrane, and the movement of DNA across the membrane into the cytoplasm.
In electroporation, an electric pulse creates pores and a temporary electric field. The electric field pulls the DNA to the more positively charged end or into the cell. When the electric field is turned off, the cell membrane reseals and traps the DNA within the cell (Carter & Shieh, 2015).
Preparing electrocompetent cells are relatively easier than making chemically competent cells. Instead of salts, chilled 10% glycerol is commonly used. Glycerol, used for extensive washing, removes remaining salts from the pellet suspension. This salt-free approach helps to prevent arcing and gives high transformation efficiency (Sharma & Schimke, 1996).
During the heat shock transformation, the heat pulse decreases the membrane potential of the competent cells, therefore lowering the potential barrier for the movement of negatively charged DNA into the cytoplasm (Panja et al., 2006).
To make chemically competent cells, pellets are usually treated with salts, for example by using CaCl2 or MgCl2. This salt treatment neutralizes the negative charges of the phospholipid bilayer and DNA, allowing DNA to move closer to the cell.
To save time and to avoid the hassle of making your own competent cells, browse and select from GoldBio’s collections of electrocompetent cells and chemically competent cells with high transformation efficiencies, which will fit your research purpose.
For more details about competent cells, find GoldBio article below:
How to Make Competent Cells
Here is a general overview about how to make competent cells.
- Prepare all solutions, including LB medium and salt solutions or glycerol solution to treat your cells.
- Streak out the E. coli strain on a plate and grow the plate overnight at 37°C.
- Collect cells from a single colony and inoculate the cells in 5 ml of LB medium overnight in a shaking incubator.
- Pipet 1 mL of the culture into 99 mL of LB Medium (1:100 dilution, no antibiotic) in a flask.
- Grow bacterial cell culture to early to mid logarithmic phase in a shaking incubator. This is typically an OD600 of 0.4.
- Centrifuge the culture at 6000 rpm for 5 minutes and decant supernatant.
- Further treat your pellet with glycerol to make them competent for electroporation or with salts for heat-shock transformation.
How to Store Competent Cells
GoldBio competent cells are shipped on dry ice. Once arrived, you must immediately store the cells in the -80°C freezer. If you store the cells at -20°C, the transformation efficiency of the cells will dramatically decrease.
How Long can Competent Cells be Stored
When stored and handled properly, GoldBio competent cells should be stable at -80°C for at least 1 year.
How Long can Competent Cells Stay on Ice
Before use, thaw and keep competent cells on ice. Incubate the thawed cells with a plasmid DNA on ice for 30 minutes prior to transformation (or a particular time suggested by your protocol) to achieve optimal transformation efficiency (Liu et al., 2014).
Why Must Competent Cells be Kept on Ice
The competent cell preparation ahead of transformation must be kept at low temperature. This low temperature helps to maintain the permeability of the cell membrane and therefore maintains high efficiency for DNA uptake.
Can You Refreeze Competent Cells?
No. Competent cells are sensitive to temperature changes, so you must avoid thawing and refreezing the cells in order to maintain the transformation efficiency of the cells.
DH10B Chemically Competent E. coli Cells (Catalog No. CC-100)
DH5-alpha Chemically Competent E. coli Cells (Catalog No. CC-101)
BL21 (DE3) Chemically Competent E. coli Cells (Catalog No. CC. 103)
DL39 (DE3) Chemically Competent E. coli Cells (Catalog No. CC-104)
DH10B Electrocompetent E. coli Cells (Catalog No. CC-200)
DH10B-Pro™ Electrocompetent E. coli Cells (Catalog No. CC-201)
DH5-alpha Electrocompetent E. coli Cells (Catalog No. CC-203)
BL21 (DE3) Electrocompetent E. coli Cells (Catalog No. CC-204)
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Carter, M., & Shieh, J. (2015, January 1). Chapter 11 - Gene Delivery Strategies (M. Carter & J. Shieh, Eds.). ScienceDirect; Academic Press. https://www.sciencedirect.com/science/article/pii/B9780128005118000113.
Chang, A., Chau, V., & Landas, J. (n.d.). Preparation of calcium competent Escherichia coli and heat-shock transformation. 1, 22–25. https://jemi.microbiology.ubc.ca/sites/default/files/Chang%20et%20al%20JEMI-methods%20Vol%201%20pg%2022-25.pdf.
Gonzales, M. F., Brooks, T., Pukatzki, S. U., & Provenzano, D. (2013). Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae. Journal of Visualized Experiments : JoVE, 80. https://doi.org/10.3791/50684.
Inoue, H., Nojima, H., & Okayama, H. (1990). High efficiency transformation of Escherichia coli with plasmids. Gene, 96(1), 23–28. https://doi.org/10.1016/0378-1119(90)90336-p.
Panja, S., Saha, S., Jana, B., & Basu, T. (2006). Role of membrane potential on artificial transformation of E. coli with plasmid DNA. Journal of Biotechnology, 127(1), 14–20. https://doi.org/10.1016/j.jbiotec.2006.06.008.
Liu, X., Liu, L., Wang, Y., Wang, X., Ma, Y., & Li, Y. (2014). The Study on the factors affecting transformation efficiency of E. coli competent cells. Pakistan Journal of Pharmaceutical Sciences, 27(3 Suppl), 679–684. https://pubmed.ncbi.nlm.nih.gov/24816699/
Sharma, R. C., & Schimke, R. T. (1996). Preparation of Electro-Competent E. coli Using Salt-Free Growth Medium. BioTechniques, 20(1), 42–44. https://doi.org/10.2144/96201bm08.