How to Troubleshoot Restriction Enzyme Based Cloning Problems

Since the early 1970s, restriction enzymes have become an important part of cloning and many other applications, including DNA mapping. Restriction enzymes are enzymes that cut DNA at or near a specific target sequence, called a restriction site.

Most of the time, as a researcher, you can’t avoid using restriction enzymes in your DNA cloning experiment. But, performing a DNA cloning experiment using restriction enzymes is not as easy as it seems. Sometimes you just can’t get those elusive white colonies on your plate, even after following the protocol strictly word for word. Therefore, in this article, we’ll look deeper into restriction enzyme based cloning, we’ll tackle some of the challenges, and give you easy ways to troubleshoot these situations.

DNA Fragment with Sticky Ends or Blunt Ends

The first step before you clone is usually choosing which restriction enzyme to use, and the type of digested products you want.

Digestion by restriction enzymes can generate either sticky ends or blunt ends on a DNA fragment. Sticky ends have short single-stranded tails (or overhangs) at each end of the DNA fragment. When those sticky DNA ends are complementary to the digested ends of your vector, you can easily ligate (join or connect) the DNA fragment and the vector together.

DNA fragment with Sticky Ends

1. Restriction Enzyme EcoRI digests a DNA fragment at a restriction site.
2. The digested DNA fragment has single-stranded overhangs (sticky ends).

Table 1. List of commonly used restriction enzymes generating sticky ends:

Blunt ends don’t have any overhangs on both ends of a digested DNA fragment. There is no requirement for blunt ends of a DNA fragment to be complementary to the other DNA ends for ligation. However, blunt ends are much harder to ligate than sticky ends.

DNA Fragment with Blunt Ends.

1. Restriction Enzyme EcoRV digests a DNA fragment at a restriction site.
2. The digested DNA fragment has no overhangs (blunt ends).

Table 2: List of some commonly used restriction enzymes generating blunt ends:

Restriction Enzyme Based Cloning Method

Restriction enzyme based cloning is dependent on two things: First, it’s dependent on the ability of the restriction enzymes to ‘cut’ both DNA fragments, that is, the fragment and the vector. Second, restriction enzyme based cloning requires the enzyme, DNA ligase, to ‘paste’ the DNA fragment into the vector. This method is relatively cheaper when compared to other cloning methods.

Restriction Enzyme Based DNA Cloning

1. Short sequences containing restriction sites are added into the 5’ ends of primers during DNA amplification by PCR.
2. Both the vector and DNA fragment are digested with restriction enzymes to create cohesive ends.
3. The vector and DNA fragment are ligated.
4. The recombinant DNA enters the host cell during transformation.

The 6 Steps of Restriction Enzyme Based Cloning

1. Preparation of a Vector

During the vector preparation step a restriction enzyme or two different restriction enzymes digest the vector at the Multiple Cloning Site (MCS) resulting in blunt ends or sticky ends. After digestion, the vector can be purified using gel electrophoresis.

You can prevent the digested vector from self-ligating during ligation of the DNA fragment and plasmid with an extra step. This step involves alkaline phosphatases, which removes phosphate groups from 5’ end of the vector.

2. Preparation of a DNA Fragment

You will need to add restriction sites to your DNA fragment.Adding those restriction sites can be achieved using PCR. Specifically, PCR primers are designed with these restriction sequences that are then incorporated into your DNA fragment.

When the PCR step is complete, digest the purified PCR products using the same restriction enzyme as the ones used during the vector preparation step. Then purify the digested products by using gel electrophoresis.

3. Ligation

To perform ligation, mix the purified vector and DNA fragment. Then add DNA ligase.

4. Transformation

After the ligation reaction is finished, select either chemically competent or electrocompetent bacteria cells to perform transformation. Transformation is the introduction of the recombinant DNA into bacteria. You can use heat shock transformation for chemically competent cells or electroporation for electrocompetent cells.

For information about the differences between the types of competent cells, take a look at our “Introduction to Competent Cells” article.

5. Selection of the colonies with the plasmid

Transformed bacteria can be cultured in media and on agar plates containing an antibiotic selection agent. Only the bacteria that carries the vector survive this selection. Take a look at our “A Quick Overview of Molecular Cloning” article for more information about how antibiotic selection and molecular cloning works.

6. Screen Clones Using Blue-White Colony Selection

Screen clones with your desired recombinant DNA by exposing the bacteria to X-Gal and IPTG (blue and white colony selection) and pick only the white clones. We have a great protocol available that details how to perform blue-white screening.

To further confirm the presence of the correct DNA fragment in the plasmid from those white clones, you can perform PCR screening, restriction digestion or DNA sequencing. After screening your desired clones, you can grow the clones using liquid media and extract the recombinant DNA for further application.

Troubleshooting for Restriction Enzyme Based Cloning

 

Below are six common cloning problems and tips to solve them:

1. Incomplete or No Digestion of PCR Product

• Check if your restriction enzyme is functional.
• Add extra nucleotides on the 5’ side of the restriction sites in your primers, and check the sequence of the restriction site to make sure there is no mutation.
• Use a long wavelength of UV light during excision from the agarose gel.
• Purify your PCR product from salt, primers, template, DNA Polymerase, and possible restriction enzyme inhibitors. One way to remove contaminants is to perform ethanol precipitation.
• Use an optimal incubation temperature and a compatible buffer for the restriction enzyme.
• Increase your incubation time for digestion.

2. Incomplete or No Digestion of Plasmid

• Check if your restriction enzyme is functional.
• Check if the activity of your restriction enzyme is blocked by DNA methylation of the target restriction site. DNA methylation is the addition of methyl groups to the DNA by DNA methyltransferase enzyme to protect the DNA. Some restriction enzymes, such as AvaI, AvaII, and ClaI, can’t recognize and cleave methylated restriction sites. If you must use these methylation sensitive restriction enzymes, choose DNA methyltransferase-free Escherichia coli strain for transformation.
• Purify your plasmid from salt and other inhibitors of restriction enzyme.
• Use an optimal incubation temperature and a compatible buffer for the restriction enzyme.
• Increase the enzyme units and your incubation time.

3. Unexpected Bands after Digestion

• Choose restriction enzymes without star activity. A star activity is a changed activity by a restriction enzyme when it cuts sequences almost similar to its restriction site.
• Decrease the enzyme units and your incubation time.
• Purify your DNA fragment and plasmid before digestion.

4. Too Many Colonies with Empty Vectors (No DNA Fragment-Vector Ligation Occurred)

• Treat your linearized plasmid with Alkaline Phosphatase prior to ligation. Alkaline Phosphatase removes phosphate groups from the 5’ ends of DNA vector, thus it prevents the vector to self-ligate during ligation.
• Increase your incubation time to make sure the digestion is complete.

5. No Colonies or Too Few Colonies

• Check if your ligase is still functional and is heat inactivated.
• Calculate the transformation efficiency of the cells. If the efficiency is too low, use commercial competent cells, and redo the transformation.
• Increase the ligation efficiency by using a different ratio of DNA fragment and plasmid.
• Increase your incubation time and use a lower temperature.
• Use the correct amount of ligase.
• Purify your DNA fragment and plasmid from ligation inhibitors.

6. High Colony Background without Plasmids

• Increase your antibiotic concentration.
• Reduce the incubation time of the plate (not more than 16 hours).
• Test if your antibiotics are functional with Disc Diffusion Method (Kirby-Bauer Test).

This article covers a quick overview about restriction enzyme based cloning, several common cloning problems that you may encounter during restriction enzyme based cloning, and some tips to solve them . Although this article only lists some of many cloning problems, we hope that these tips can be a good start to conquer your cloning experiments.

REFERENCES

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Bhagwat, A. S. (1995). [6] - Restriction Enzymes: Properties and Use. In R. Wu (Ed.), Recombinant DNA Methodology II (pp. 67-92). Boston: Academic Press.

Carter, M., & Shieh, J. C. (2010). Chapter 9 - Molecular Cloning and Recombinant DNA Technology. In M. Carter & J. C. Shieh (Eds.), Guide to Research Techniques in Neuroscience (pp. 207-227). New York: Academic Press.

Celie, P. H. N., Parret, A. H. A., & Perrakis, A. (2016). Recombinant cloning strategies for protein expression. Current Opinion in Structural Biology, 38, 145-154. doi:https://doi.org/10.1016/j.sbi.2016.06.010

Cohen, S. N., Chang, A. C. Y., Boyer, H. W., & Helling, R. B. (1973). Construction of Biologically Functional Bacterial Plasmids In Vitro. Proceedings of the National Academy of Sciences, 70(11), 3240. doi:10.1073/pnas.70.11.3240

Lessard, J. C. (2013). Chapter Seven - Molecular Cloning. In J. Lorsch (Ed.), Methods in Enzymology (Vol. 529, pp. 85-98): Academic Press.

Matsumura, I. (2015). Why Johnny can't clone: Common pitfalls and not so common solutions. BioTechniques, 59(3), IV-XIII. doi:10.2144/000114324.

McClelland, M., & Nelson, M. (1988). The effect of site-specific DNA methylation on restriction endonucleases and DNA modification methyltransferases — a review. Gene, 74(1), 291-304. doi:https://doi.org/10.1016/0378-1119(88)90305-8.