Bacterial Transformation with Plasmid DNA: A How-To Guide

Bacterial transformation with DNA is a common molecular biology technique that is used to amplify small, circular pieces of DNA called plasmids. Briefly, researchers will clone a small copy of some gene into a plasmid, introduce that plasmid into a bacterial strain, and then use those bacteria to replicate that piece of DNA into large quantities.

The video to the right gives a brief visual explanation of transformation. In the video, the transformed DNA is a non-circular fragment. However, in the lab, we generally use circularized plasmids. Either way, the concept of the uptake of DNA by bacteria is the same.

A bacterial transformation is a relatively easy and straightforward technique. In order to perform a transformation, you will need the following:

• Bucket of ice
• Polypropylene tubes
• Plasmid DNA in water or TE buffer
• Transformation competent Escherichia coli (E. coli)
• Hot water bath (42 degrees Celsius)
• S.O.C. Medium
• LB agar plates with the correct antibiotic selection marker
• Incubator / shaker for bacteria (37 degrees Celsius)

Researchers have found that certain bacterial strains are able to naturally take up pieces of DNA. The technique of bacterial transformation artificially harnesses that ability for the purposes of research. “Competent” cells are bacteria that are able to take up plasmids under specific conditions. In the lab, we are able to induce artificial competence for strains of bacteria that do not normally take up plasmids, but are easily grown in laboratory bacterial cultures.

The most common “competent” bacterial strain is DH5α E. coli. These bacteria are not normally competent to take up DNA. However, when E. coli are incubated on ice in the presence of a divalent cation (e.g. Magnesium, Calcium), the cell wall that envelops the bacteria becomes permeable to DNA. Although the exact mechanism by which the DNA crosses the cell wall is unknown, researchers have speculated that the divalent cation may coordinate the negative charges on the DNA, thereby shielding the DNA from negative charges of the cell wall, and allowing it to pass into the bacterium.

Plasmids are small circular pieces of DNA that are commonly used in molecular biology to study specific genes. The open reading frame, among other sequences, can be cloned into a plasmid so that it can be used in experiments.

Typically, a plasmid has five main components. They are as follows:

• Gene insert
• Promoter to drive gene expression
• Origin of replication
• Antibiotic resistance marker.
• Mammalian selection marker.

The insert is the cloned gene under study. The “promoter” is a short sequence of DNA that controls expression of that gene when it is placed into mammalian cells. An origin of replication is required so that bacteria can replicate the plasmid. Finally, the antibiotic resistance marker is required so that contaminating bacteria do not affect the culture when E. coli are grown in a lab. An antibiotic is added to the medium so that only bacteria which have the plasmid are able to grow.

1. All competent DH5α E. coli should be kept at -80 degrees Celsius.
2. Remove the E. coli from the freezer and allow them to thaw on ice. This can take as long as fifteen minutes.
3. Once the E. coli are thawed, take 50 microliters (ul) of the bacteria and place it into a polypropylene tube. Use one tube for each plasmid you want to transform.
4. Add 1-5 nanograms of plasmid DNA to each one of your polypropylene tubes. Do not pipette up and down or vortex the tube. Just pipette the DNA directly into the bacteria. (If you are using a ligation reaction, use up to 10 ul of the ligation mixture)
5. Let the DNA/bacteria incubate on ice for 45 minutes. Make sure the tubes are nearly covered in ice. It is important to keep the mixture cold at this point.
6. During the incubation, make sure that you have a water bath set to 42 degrees Celsius.
7. After the incubation, heat-shock the bacteria in the polypropylene tubes by placing them in the water bath for 45 seconds. (Alternatively, I have successfully heat shocked DH5α E. coli for 90 seconds at 37 degrees Celsius).
8. Immediately after you remove the bacteria from the water bath, place 700 ul of S.O.C. medium into the polypropylene tubes. It is important to use sterile technique at this point. Be sure to heat flash pipette tips and contain openings.
9. After adding the S.O.C., place the polypropylene tubes in the incubator/shaker for 45 minutes at 37 degrees Celsius and 225 rpm. Do not include an antibiotic at this point.
10. While the bacteria are shaking, remove LB agar plates from the cold-room or fridge, and let them warm to room temperature. Make sure the antibiotic of the plate corresponds to the resistance marker of your plasmid.
11. Remove the bacteria from the incubator and place 150 ul of the SOC medium onto one of the LB-agar plates. 150 ul is a volume that works well for me in my lab. You may want to try two different volumes. For example, add 150 ul to one plate and add 300 to another plate. Using two volumes will ensure that colonies are present and evenly spaced the following morning.
12. Use a cell spreader to spread the S.O.C./bacteria mixture onto the surface of the LB-agar plate.
13. Let dry for about 5-10 minutes.
14. Replace the lid onto the LB-agar plates and place the plates, inverted, into the incubator.
15. Let the plates incubate overnight, and check on them the next morning to harvest your colonies.

What do you do if you come to the lab the next morning and there are no colonies?

• First, check to make sure the bacteria you used were not too old and that it hadn’t been thawed before. (Be sure to always check beforehand, too, so that this is not a recurring problem).
• If you used new bacteria, you may want to run your DNA on an agarose gel to make sure it hasn’t been degraded by contaminating nucleases.
• Make sure the water bath is at the correct temperature. Heat shocking the bacteria is very important, as this step facilitates the uptake of DNA by bacteria. Sometimes, although a water bath is set to the right temperature, the water will not actually be the right temperature. Use multiple thermometers to check.
• Make sure the LB-agar plates are not out-of-date.
• Check to see that the SOC medium is not contaminated with bacteria.

One of the major reasons that researchers transform bacteria is to amplify enough DNA for restriction digestions and cloning. Restriction digestions are enzymatic reactions where nucleases cleave specific DNA sequences. However, selecting the right strain of bacteria is critical if you are transforming for this purpose. Some strains of E. coli will methylate specific sequences of DNA and these methylations will block the ability of restriction enzymes to cleave DNA at that location. To resolve this issue, researchers have created E. coli strains that are defective in DNA methylation. These strains, usually marked as dam-/-, dcm-/-, will help you clone your DNA if you need to use a methylation-sensitive restriction enzyme.

Bacterial transformation with DNA is a common, but very important, tool for molecular biology. Mastering this technique can save a lot of time and frustration, as well as avoid delays in starting real experiments.