Molecular Biology. A Practical Guide - Velikov V.A. 2013

Bacterial Transformation
Transformation of E. coli

To transform E. coli, competent Cells are combined with purified plasmid DNA or a "ligation mixture". The DNA is added to the cells and incubated on ice for a short period to allow for DNA sorption onto The Cell surface. Next, a brief "heat Shock" is applied at 42°C, causing the DNA to enter the E. coli cells due to a sharp increase in membrane fluidity and local phase inversions. Nutrient broth is then added, and the Bacteria are plated onto an Agar growth medium containing an antibiotic. Driven by the expression of plasmid antibiotic-resistance genes, the transformed cells acquire The ability to thrive on antibiotic-supplemented media. Within 1–2 days, clearly visible colonies (approx. 10⁴–10⁵ cells) emerge on the solid growth medium—derived exclusively from the cells that successfully took up the plasmid, i.e., the transformant clones.

Not all transformants are recombinants; cells can also be transformed with the original ("empty") vector unless special precautions are taken to prevent this. Molecular biologists, for instance, typically use two different restriction Enzymes for DNA Digestion or treat the vector DNA with alkaline phosphatase to prevent self-ligation of the vector. Recombinant clones can be distinguished from empty vector colonies after plating on nutrient agar using a Procedure known as "blue-white screening" (see Appendices 5, 6, 14).

The transformation procedure outlined below is standard. Prior to the advent of electroporators, it was widely used in molecular biological and biotechnological research and remains in practice today.

Materials and Equipment

Competent calcium-chloride-treated E. coli XL1-Blue cells, plasmid pBR322 DNA solution (0.5 µg/mL), micro-incubator.

Solutions

- SOC medium. Per 100 mL: add 5 mL of 20% glucose to SOB medium (Exercise 8.1).

- 0.5 M ß-mercaptoethanol.

Procedure

1. Prepare a plasmid pBR322 DNA solution at a concentration of 5 ng/mL. To do this, take 1 µL from the concentrated 100x stock solution (0.5 µg/mL) and add it to 100 µL of deionized Water.

2. Thaw on ice 0.5 M ß-mercaptoethanol (an antioxidant and disulfide bond reducing agent) and an aliquot of competent cells.

3. To a 100 µL aliquot of competent cells, add 4 µL of 0.5 M ß-mercaptoethanol and 10–50 pg (2–10 µL) of the prepared pBR322 DNA solution.

4. Leave the tube at 0°C for 20–30 minutes without shaking to allow the DNA to settle onto the cell surface.

5. Perform a heat shock at 42°C for 30 s ^a in a water bath or micro-incubator. Immediately afterwards, place the tube on ice for 1–2 minutes.

6. Add 400 µL of SOC medium and incubate on a shaker at 37⁰C for 30 minutes.

7. Plate a 50 µL aliquot of the cell suspension onto a 2YT agar plate containing 100 µg/mL ampicillin ^б.

8. Evaluate transformation efficiency the following day. If N colonies grow on the Petri dish, the transformation efficiency is N x 10⁶ colonies per 1 µg of DNA ^в.

Notes

^a Some protocols recommend a longer heat shock (up to 3 minutes).

^б Plating on agar media is performed as follows. Sterilize a Glass spreader in a gas burner flame and let it cool. Place an aliquot of the cell suspension onto The surface of pre-dried nutrient agar. Spread the suspension evenly across the agar surface using the glass spreader until all liquid is completely absorbed.

^в If maximizing the colony yield on 2YT agar is required—such as when constructing a genomic library—plate 10–50 µL of the suspension to determine the titer, and store the remaining cells overnight at 0°C without loss of titer. The next day, calculate the required plating volume and spread the bacteria onto selective agar. Maximum transformation efficiency can reach 10⁹–10¹⁰ transformant cells per 1 µg of plasmid DNA.