DH5a competent cells are a genetically engineered Escherichia coli (E. coli) strain designed for high-efficiency DNA transformation. DH5a is a go-to strain for DNA cloning, plasmid propagation, library preparation, and more.

What if you need to do some cloning to create and amplify new DNA plasmids? While Escherichia coli BL21 strains excel at producing recombinant proteins, it helps to have a strain suited for cloning.

Fortunately, bacteria are amazing molecular factories. They are incredibly useful at generating and scaling up custom biomolecules for a variety of research and biotechnology applications.

In this article, we’ll dive into an E. coli strain that is a great choice for DNA cloning: DH5a.

DH5a competent cells are a genetically engineered Escherichia coli (E. coli) strain designed for high-efficiency DNA transformation. DH5a is a go-to strain for DNA cloning, plasmid propagation, library preparation, and more.

Special Features of DH5 alpha cells

Why is DH5a such a good E. coli strain for DNA cloning? DH5a cells have point mutations in the genes that encode for the enzymes RecA and EndA (Yale University, 2020).

RecA is a recombinase, or an enzyme that catalyzes the process of homologous recombination between different DNA molecules. In the context of DNA cloning in bacteria, this would mean RecA could integrate your plasmid into the bacterial genome, or append multiple plasmid molecules together.

The RecA mutation inactivates this enzyme, keeping your plasmid as a single copy and out of the bacterial genome where it’s available for you to harvest when you’re done with your cloning step.

EndA is a nuclease that cuts up double-stranded DNA. The EndA mutation also inactivates this enzyme, meaning that EndA can’t cleave your plasmid in the bacterial cells, increasing the overall DNA yield (Figure 1).

Figure 1. Mutated EndA nuclease enables DH5a to generate higher plasmid DNA concentrations (right).

Another way of thinking about the EndA mutation is making cookies. If your helper in the kitchen keeps eating the cookie dough before you can bake it, then you will end up with less cookies. The EndA mutation is like tying your helper’s hands behind their back so that they can’t eat the cookie dough before you bake it.

What are GB5-a competent cells?

The DH5a strain has that name because it was the 5^th strain engineered by the scientist Douglas Hanahan. As it turns out, that 5^th strain ended up being the best at producing DNA plasmids, so that is why it is commonly used today (Hanahan, 1985).

GoldBio has its own version of DH5a cells. These are called GB5-a^TM cells, and they have the same mutations that we just discussed. Importantly, GB5-a^TM E. coli cells are equivalent to DH5a. So, if your protocol calls for DH5a, you can confidently use GB5-a^TM cells knowing that you are using the right strain at a great price.

Electrocompetent vs chemically competent cells

GoldBio sells two different kinds of GB5-a^TM: electrocompetent and chemically competent. The only difference between these cells is how you transform DNA into them.

Electrocompetent cells use electroporation to transfer DNA into the cells, whereas chemically competent GB5-a^TM cells use a mild heat shock for the same purpose.

If you want more details about the differences between electrocompetent and chemically competent cells, check out this article.

One important difference to highlight is that transformation efficiencies tend to be higher for electroporation compared to chemical transformation (Aune & Aachmann, 2010).

In the next section we’ll discuss different applications for DH5a cells. Your intended application may dictate which type of DH5a strain you should use.

Transformation efficiency is not critical for standard cloning procedures, so with most of your regular cloning needs it won’t really matter whether you use electrocompetent or chemically competent cells.

However, transformation efficiency can be critical for more advanced cloning applications, such as library preparations. In these cases, you would probably want to use electrocompetent cells to maximize transformation efficiency.

There are several DNA cloning applications that DH5a cells are useful for, including:

• standard cloning
• blue-white screening
• DNA library construction

 

Standard DNA cloning

These are your typical DNA cloning techniques. For example, you’re working with a new protein and you want to create an expression plasmid for it. You would use DH5a cells to help create and amplify this new plasmid, and make a lot of the DNA. Then you would use BL21 cells to express the protein.

Another typical use is point-mutagenesis. Sometimes it is important to tease apart the importance of a single amino acid for a protein’s structure or function. It’s common practice to mutate single residues to a different amino acid for these types of studies, and one way to generate these plasmids is to clone in the mutation that you want.

For standard DNA cloning techniques, transformation efficiency is not crucial. This is because there is just one correct clone that you are trying to get, so even if your transformation is mediocre, you’ll probably still be able to determine if your cloning step worked, or not.

So, it is typically not critical whether you use electrocompetent or chemically competent cells for standard DNA cloning procedures.

Blue-white screening

Blue-white screening is a convenient way to determine if your cloning step worked just by looking at the color of the bacterial colonies.

When doing the types of cloning we just described in the previous section, to confirm that your cloning step worked you would pick a bacterial colony, grow more cells from the colony, lyse the cells and isolate the plasmid DNA, then either cut out the DNA and run it on an agarose gel, or send the DNA for sequencing (Figure 2).

Figure 2. Cutting the gene of interest out of the plasmid is one way to confirm that your cloning step worked.

If that sounds like a lot of work, that’s because it is. Especially if you’re working on multiple clones. And usually, you’ll process a few bacterial colonies for each DNA construct that you’re trying to make to ensure that at least one of them is correct. Because, nothing is worse than processing several samples for a couple of days only to find that none of them have the plasmid you were trying to clone. (Don’t ask me how I know …)

Wouldn’t it be fantastic if you could just look at the bacterial colonies instead and instantly know if your cloning worked?

That’s where blue-white screening comes in. Check out this article if you want more details about how exactly blue-white screening works. But the key takeaway is that in compatible plasmids such as pUC19, if your gene was inserted successfully, the bacteria will look white. Whereas, if the gene wasn’t inserted the bacteria are blue. So that tells you to only pick the white colonies for further confirmation of your cloning step (Figure 3).

Figure 3. Blue bacterial colonies have no gene inserted whereas white colonies do, so the white colonies are the ones you want to pick to verify your gene was inserted.

DNA library construction

In the previous two sections we were discussing scenarios where you’re only cloning one thing at a time. Sometimes though, you’re trying to create thousands or even millions of different plasmids in parallel. These large collections of plasmids are called libraries, and can be generated for a variety of different reasons. If we think about this DNA library like an actual library, then each individual plasmid would be like a single book.

There are wide range of DNA libraries that scientists construct, including genomic DNA, complementary DNA, mutagenesis, and small guide RNA libraries (Kadooka & Oka, 2024; National Library of Medicine, 2025; Nishiga et al, 2021; Oehlmann & Rebelein, 2025; Soares et al, 1994).

When creating any of these libraries, however, it makes a big difference if we generate 10 plasmids or 10,000 plasmids because those additional 9,990 plasmids represent a variety of different plasmids that will enrich our experiment (Figure 4).

Figure 4. When cloning a single plasmid (left) transformational efficiency isn’t crucial as long as you get a single clone with the plasmid you want. When constructing plasmid libraries (right) low transformational efficiency will limit the diversity of your library.

These numbers are not exact, of course, I’m just illustrating a point. But, if you’re constructing plasmid libraries with DH5a cells, you would likely want to use GB5-a^TM electrocompetent cells to maximize your transformation efficiency.

Another type of bacteria strain, DH10B has even higher efficiency than either version of DH5a cells. So, if you’re constructing a plasmid library our GB10B-Pro^TM cells are the optimal choice. See this article for more details about GB10B cells.

In summary, DH5a cells are a great choice for DNA cloning, and GB5-a^TM give you all the benefits of this strain at a great price. If you’re ready to get going, check out our reliable products below to help jumpstart your DNA cloning in the lab. Alternatively, if you are still at the learning and planning stage, check out some of our related resources to learn more about competent cells, DNA plasmids, and blue-white screening.

 

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Cloning competent cells Competent CellsDh5 alpha Simon Currie

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