Overview:

Small-scale plasmid isolation, called plasmid miniprep, requires a small amount of bacterial culture. Normally, a 2 – 5 ml culture provides sufficient cells that can be processed for plasmid isolation using the miniprep protocol. Culture can be prepared by inoculating 3 ml antibiotic-containing growth medium with a single colony and growing it overnight at 37C with shaking.

Here we have taken an example of preparing liquid culture from a colony of E. coli DH5α, transformed with the pEGFP plasmid. The pEGFP plasmid contains a kanamycin resistance gene, therefore, requires kanamycin for the selection of plasmid-containing bacteria. If your plasmid carries another antibiotic-resistant gene, add the respective antibiotic to the culture medium.

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REQUIREMENTS

Reagents and solutions
♦ LB medium [see Preparation of Luria-Bertani (LB), Miller Broth]
♦ Kanamycin (Stock conc. 50 mg/ml) [See Preparation of Stock Solution of Kanamycin (50 mg/ml)]

Equipment and disposables
♦ Falcon®14mL Round Bottom polypropylene tube with Snap Cap (Cat No. #352059)/ 25-ml conical flask with cotton plug (autoclaved)
♦ Benchtop Shaking Incubator
♦ Bunsen burner
♦ Clean workbench
♦ Autoclaved toothpick/Pipette tips/Inoculation loop

OBJECTIVE
Growing liquid culture of E. coli DH5α harboring pEGFP plasmid for miniprep

STARTING MATERIAL
Bacterial colony on antibiotic-containing LB-Agar plate (in our case on Kanamycin LAB-agar plate)

PROCEDURE

Prior to start
Set the shaking incubator at 37°C.

General instructions
◊ The best is to use the microbial hood (or Laminar flow hood) to perform all operations aseptically. If it is not possible to use a microbial hood, perform all microbiological operations close to the flame of the bunsen burner in a clean place wiped with 70% ethanol.
◊ Do all operations aseptically and use sterile material and reagents. All operation that involves the transfer of medium from one bottle to another or the transfer of a colony to the medium should be done quickly to reduce the risk of contamination.
◊ Before starting your work, clean your hands with soap and wipe them with 70% ethanol.
◊ Use appropriate personal protective equipment (lab coat, gloves, goggles, etc) for your safety and follow your institute’s guidelines. You must read the material safety data sheet (MSDS) before working with any chemical.

Step 1: Prepare LB medium with antibiotics
♦ Transfer 3 ml LB medium aseptically to a polypropylene tube.
♦ Add 3 µl of kanamycin stock solution (50 mg/ml). The final concentration of kanamycin will be 50 µg/ml.

Note:
◊ A single colony can be inoculated in a 2 – 10 ml culture volume. Since miniprep needs 1 – 3 ml culture, inoculating, a 3 ml culture medium is sufficient. Depending on the volume of the culture medium, you should choose appropriately sized vessels.
◊ Disposable plastic tubes with Snap Cap are a good choice for culture vessels. These tubes are available in a ready-to-use form (sterile), easy to cap and provide good aeration, and cheap. These tubes can be discarded after use, therefore, no effort is required for cleaning and preparing them for the next use. Conical flasks and glass test tubes can also be used for culturing bacteria.
◊ Depending on how many colonies you want to inoculate, prepare the same number of tubes/flasks. If you are screening for the presence of an insert in a plasmid, you need to inoculate many colonies in separate polypropylene tubes. For example, if you need to inoculate 10 colonies, you must prepare 10 polypropylene tubes with the culture medium. In this case, you need a 30 ml culture medium. Take 30 ml culture medium, add 30 µl kanamycin, and distribute 3 ml in each polypropylene tube.

Tip
◊ You can either pour or use a sterile pipette to transfer the liquid medium into the tube. Since Falcon polypropylene tubes have markings, pouring is more convenient and quick if you have many colonies to inoculate. However, It can also increase the risk of contamination.

Precaution:
◊ Whenever you open the media bottle, show the mouth of the bottle to the flame.

Step 2: Inoculate the culture medium with the bacterial colony
♦ Touch the surface of a bacterial colony with a sterile toothpick or pipette tip.
♦ Drop it into the antibiotic-containing LB medium.

Note: 
◊ Toothpicks and pipette tips are convenient to use. If you are using a metallic inoculation loop, red-heat it and then wait till it has cooled to normal temperature. Touch the colony and put it in the medium and shake the inoculation loop to dislodge bacterial cells in the culture medium. Disposable sterile inoculation loops are also available commercially and are convenient to use for inoculation. They can be used once and should be discarded after use.

Precautions:
◊ Don’t inoculate the culture medium directly from a glycerol stock. This can cause low yields and unpredictable results.
◊ Make sure that at least some bacterial cells stick to the toothpick/pipette tip while picking up the colony from the LB-Agar plate.

Step 3: Grow the culture overnight (12 – 16 h) at 37°C with vigorous shaking.
♦ Set the culture tube/flask in the shaker incubator. Use an appropriate inclined (30° – 45°) angle if you are using a polypropylene tube to ensure good shaking.
♦ Set the speed to 200 – 300 rpm and start the shaker. Incubate for 12 – 16 h.

Precautions:
◊ Don’t grow more than 16h.
◊  Make sure that the aeration of good as good aeration is required for E. coli growth.

Step 4: Take the culture out from the incubator the next morning. Culture is ready for plasmid isolation.

Note:
◊ It is always good to start the isolation process immediately or on the same day. Culture can be stored for 2 – 3 days at 4°C. Longer storage may cause low plasmid yield.