Troubleshooting: Bacterial Transformation

Bacterial transformation is a routine procedure in molecular biology laboratories. After transforming a ligation reaction into Escherichia coli competent cells and plating the cells, you only have to wait for your plate to grow a decent number of colonies on the following day.

But, oftentimes you come across some common problems on your plate, such as no colonies, satellite colonies, or too many colonies. This article provides you with some factors affecting successful bacterial transformation and a quick troubleshooting guide to solve your problems after transformation.

What are Some Factors Affecting Successful Bacterial Transformation?

1.Transformation Efficiencies of the Competent Cells

Competent cells with low transformation efficiencies cause few or no colonies growing on the plate. To calculate the transformation efficiency, use an uncut plasmid with a known concentration, such as pUC19, to transform your competent cells.

How to calculate transformation efficiency

Transformation efficiency is the number of colony forming units produced by transforming 1 µg of plasmid DNA into a given volume of competent cells.

As an example, you transformed 1 µl of (10 pg/µl) pUC19 into 25 µl of GoldBio GB10B™ chemically competent cells.

You then added 975 µl of Recovery Medium into your tube. You diluted 10 µl of this in 990 µl of Recovery Medium and plated 50 µl of the diluted medium.

On the next day, you counted 250 colonies on your plate.

Based on this information, calculate:

• Colonies = 250
• µg of DNA = 0.00001 (or 10 pg = 0.00001 ug)
• Dilution = 10/1000 x 50/1000 = 0.0005

Therefore, the transformation efficiency of your competent cells:

TE = 250/0.00001/0.0005 = 5.0 × 10¹⁰

For a small plasmid, such as pUC19, 5.0 × 10¹⁰ cfu/μg is a relatively high TE. Therefore, these competent cells are highly efficient.

To learn more about how to calculate the transformation efficiency of your competent cells, watch GoldBio video below:

2.Plasmid

The plasmid used in transformation may affect the transformation efficiency. As an example, the efficiency of transformation using a large size plasmid is typically lower than the efficiency using a small size plasmid. To transform a large plasmid, choosing the right competent cells and electroporation method can help improve the efficiency of the transformation.

Another factor to consider, check the concentration of DNA to use as recommended by your protocol. Based on the protocol for GoldBio GB10B™ chemically competent cells, the recommended amounts of DNA used for the transformation are between 1 pg - 100 ng of DNA. Therefore, you can use 1 µl of the ligation reaction containing at least 1 pg of DNA for transforming these cells.

3.Growth Medium

Transformed bacteria undergo a heat-shock step or an electric pulse to create temporary pores in their cell wall. Therefore, they can easily take up plasmid DNA. To recover from this stress, the cells need to live and grow in a nutrient-rich medium, such as SOC medium.

To find out more about these special media, read GoldBio’s article below

A Quick Overview of SOC Medium & Competent Cell Recovery Medium

4.Temperature

Temperature plays a key role during the bacterial transformation, particularly for transforming chemically competent cells.

As an example, for GoldBio GB10B™ chemically competent cells, the heat-shock step requires sequential treatments: incubation at 0°C for 30 minutes, followed by incubation at 42°C for 45 seconds, and then back at 0°C for 2 minutes. Any incorrect step in this process can affect the result of transformation.

Directly after transformation, the transformed cells need a warm temperature of 37°C to grow optimally. One method to do this is by using a shaking incubator. This type of incubator also distributes nutrients evenly to all the cells in the growth medium.

5.Antibiotic Selection

The antibiotic in your plate affects the number of colonies to grow on the plate after transformation. Using the wrong antibiotic causes no colonies growing on the plate. Whereas, using an antibiotic concentration, which is too low, results in bacterial lawn. A bacterial lawn is the appearance of bacterial colonies on your plate forming a uniform layer.

To make sure you use the correct antibiotic, check the selectable marker in your plasmid. A selectable marker is typically antibiotic resistance gene. As an example, if your plasmid contains an ampicillin resistance gene, use ampicillin in your plate to weed out your untransformed cells. Therefore, only transformed cells grow on the plate.

Regardless of the antibiotic you use, keep in mind that incubating your plate for more than 16 hours can cause the growth of satellite colonies.

To troubleshoot satellite colonies, find GoldBio article below:

Troubleshooting: satellite colonies

Troubleshooting Guide for Bacterial Transformation

1.Test the Transformation Efficiency of the Competent Cells

Include a control plate in your experiment and calculate the transformation efficiency. You can use our calculator to calculate this number. To allow efficient transformation, use commercially available competent cells, such as GoldBio Competent Cells.

2.Check the Transformation Protocol

Performing a wrong step during your transformation can affect success. Therefore, check if you did all transformation steps correctly.

3.Check the Antibiotics

Make sure you used the correct antibiotic with the recommended concentration to select your transformed cells. In addition, before adding, make sure your antibiotic is not too old and the temperature of your media is not too hot.

4.Test the Growth Media

Use a special medium to grow transformed cells, such as SOC medium or GoldBio’s Competent Cells Recovery Medium. In addition, test your plate by streaking E. coli cells.

Related Products

GoldBio’s Competent Cells

GB10B™ Chemically Competent E. coli Cells (Catalog No. CC-100)

GB5-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)

GB10B™ Electrocompetent E. coli Cells (Catalog No. CC-200)

GB10B-Pro™ Electrocompetent E. coli Cells (Catalog No. CC-201)

GB5-alpha™ Electrocompetent E. coli Cells (Catalog No. CC-203)

BL21 (DE3) Electrocompetent E. coli Cells (Catalog No. CC-204)

Popular Antibiotics Products

Ampicillin (Sodium), USP Grade (Catalog No. A-301)

Ampicillin (Sodium) EZ Pak™ for 100 mg/mL Solution (Catalog No. A-301-EZ)

References

‌Gooch, J. W. (2011). Bacterial Lawn. Encyclopedic Dictionary of Polymers, 877–877. https://doi.org/10.1007/978-1-4419-6247-8_13222.

Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology, 166(4), 557–580. https://doi.org/10.1016/s0022-2836(83)80284-8.

Medaney, F., Dimitriu, T., Ellis, R. J., & Raymond, B. (2016). Live to cheat another day: bacterial dormancy facilitates the social exploitation of β-lactamases. The ISME journal, 10(3), 778.

Ohse, M., Takahashi, K., Kadowaki, Y., & Kusaoke, H. (1995). Effects of plasmid DNA sizes and several other factors on transformation of Bacillus subtilis ISW1214 with plasmid DNA by electroporation. Bioscience, Biotechnology, and Biochemistry, 59(8), 1433–1437. https://doi.org/10.1271/bbb.59.1433.

Rahimzadeh, M., Sadeghizadeh, M., Najafi, F., Arab, S., & Mobasheri, H. (2016). Impact of heat shock step on bacterial transformation efficiency. Molecular Biology Research Communications, 5(4), 257–261. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC53264...

Rolinson, G., Macdonald, A., & Wilson, D. (1977). Bactericidal action of β-lactam antibiotics on Escherichia coli with particular reference to ampicillin and amoxycillin. Journal of Antimicrobial Chemotherapy, 3(6), 541-553.

Yurtsev, E. A., Chao, H. X., Datta, M. S., Artemova, T., & Gore, J. (2013). Bacterial cheating drives the population dynamics of cooperative antibiotic resistance plasmids. Molecular Systems Biology, 9(1), 683. doi:10.1038/msb.2013.39