- A small-scale plasmid isolation procedure, the miniprep, yields a sufficient amount of plasmid for the screening of clones and DNA sequencing. Once a clone is confirmed for the presence of insert with the right sequence, a large amount of plasmid can be prepared by midiprep or maxiprep.
- Miniprep requires a small amount of culture of the plasmid-containing bacterial cells. Most often a single colony from the LB-agar plate is inoculated in a liquid medium. Culture is grown at 37°C in a shaker incubator overnight (12- 16 h). Grown culture corresponds to the late log phase/early stationary phase of bacterial growth and is characterized by the low content of RNA. At this stage, the grown culture has a density of 3 – 4 × 109 cells/ml and OD600 ∼ 0.8.
- Sometimes, a well-grown colony from the LB-agar plate can directly be utilized for plasmid isolation. A well-grown colony on the LB-agar plate is prepared by streaking a colony in a small area (0.5 – 1 cm long). This is more convenient when you need to screen a large number of colonies. It is also cost-effective, requires fewer reagents and no handling of culture flask.
- An antibiotic should be present at all stages of culture growth. The choice of antibiotic depends on the antibiotic-resistant gene carried by the plasmid. In absence of the antibiotic, dividing cells can lose the plasmid, resulting in low plasmid yield.
1. 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 selection of plasmid-containing bacteria.
2. If your plasmid carries another antibiotic-resistant gene, add the respective antibiotic in the culture medium.
> LB medium
> Kanamycin (Stock conc. 50 mg/ml)
> Falcon® 14mL Round Bottom polypropylene tube with Snap Cap (Cat No. #352059)/ 25-ml conical flask with cotton plug (autoclaved)
Equipment and disposables
> Bunsen burner
> Clean workbench
> Autoclaved toothpick/Pipette tips/Inoculation loop
Growing liquid culture of E. coli DH5α harboring pEGFP plasmid for miniprep
Bacterial colony on antibiotic-containing LB-Agar plate
Prior to start
Set the shaking incubator at 37°C.
> Perform all microbiological operations close to the flame of the bunsen burner in a clean place wiped with 70% ethanol or within the microbial hood.
> Do all operations aseptically and use sterile material and reagents. All operation which involves opening of a media bottle should be done quickly to reduce the risk of contamination. Before starting your work, clean your hands with soap. You can also wear gloves. Wipe your hand with 70% ethanol.
Step 1: Prepare LB medium with antibiotics
> Transfer 3 ml LB medium aseptically to polypropylene tube (Falcon, Cat No. #352059).
> Add 3 µl of antibiotics stock solution of kanamycin (50 mg/ml). The final concentration of kanamycin will be 50 µg/ml.
1. A single colony can be inoculated in a 2 – 10 ml culture volume. Since miniprep needs 1 – 3 ml culture, inoculating 3 ml culture medium is sufficient. Depending on the volume of the culture medium, you should choose appropriately sized vessels.
2. Disposable plastic tubes with Snap Cap is a good choice for culture vessels. These tubes are available in ready to use form (sterile), easy to cap and provide good aeration, and are 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.
3. Depending on how many colonies you want to inoculate, prepare the same number of 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.
You can either pour or use a sterile pipette to transfer the liquid medium into the tube. Since Falcon polypropylene tubes (Cat No. #352059) have markings, pouring is more convenient and quick if you have many colonies to inoculate. However, It can also increase the risk of contamination.
Whenever you open the media bottle, show the mouth of the bottle to the flame.
Step 2: Inoculate 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.
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 inoculation loops are sterile and ready to use. They can be used once and discarded after use.
1. Don’t inoculate culture medium directly from a glycerol stock. This can cause low yield and unpredictable results.
2. 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 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 200 – 300 rpm and start the shaker. Incubate for 12 – 16 h.
Don’t grow more than 16h.
Step 4: Take out the culture the next morning. Culture is ready for plasmid isolation.
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.