Protocol – Growing Large Volume of Liquid Culture of Escherichia coli for Large-Scale Plasmid Isolation

OVERVIEW

  • Large-scale isolation of plasmid requires a large volume of E. coli culture. 
  • Large-scale plasmid isolation procedures are termed midiprep (25 – 50 ml starting culture volume) and maxiprep (100 – 500 ml starting culture volume).
  • A starter culture is initially prepared by inoculating a colony in a small volume (2 – 10 ml) of culture medium.
  • Large culture volume is prepared by diluting starter culture in a ratio of 1: 100 to 1: 1000 in the growth medium.

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

REQUIREMENTS

Reagents and solutions
LB medium
Ampicillin (Stock conc 50 mg/ml)
A 25-ml conical flask with cotton plug (autoclaved)/Falcon polypropylene tubes (Cat No. #352059)
A 500-ml conical flask with cotton plug (autoclaved)

Equipment and disposables
Bunsen burner
Clean workbench
Autoclaved toothpicks/Pipette tips/Inoculation loop

Objective
Growing large volume of culture (100 ml) of E. coli harboring pcDNA plasmid for large scale plasmid isolation

Starting material
Bacterial colonies on ampicillin-containing LB-Agar plate

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

General precautions:
◊ Do all operations aseptically and use sterile material and reagents. If you do not have a laminar flow hood, perform all microbiological operations close to the flame of Bunsen burner in a clean place, wiped with 70% ethanol.

◊ All operations which involve opening of media bottles should be done quickly to reduce the risk of contamination.
◊ Before starting your work, clean your hands with soap.
◊ Whenever you open a media bottle, show the mouth of the bottle to the flame.

PROCEDURE

A. Preparing Starter Culture

Step 1: Prepare 5 ml LB broth with appropriate antibiotic (ampicillin in case of pcDNA)
◊ Transfer 5 ml LB medium aseptically to a 25-ml conical flask. 
◊ Add 10 µl of ampicillin antibiotic stock solution (50 mg/ml). Swirl the flask. 

Note:
The final concentration of ampicillin will be 100 µg/ml.

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

Precautions:
1. Don’t inoculate culture medium directly from 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 for 8 – 12 h at 37°C with vigorous shaking.
◊ Set the flask in a shaker incubator.
◊ Set the speed 200 – 300 rpm and start the shaker. Incubate for 8 – 12 h.

Note:
Incubation for 8 h is sufficient to see turbidity. At this growth stage, the culture will be in the long phase of the growth curve, which represents exponential growing cells. When these cells are diluted, they will maintain their exponential growth.

Tip:
A good way to prepare starter culture is to inoculate a colony in the morning. Starter culture will be ready in the evening.

B. Preparation of large volume of cultures by diluting starter culture in growth medium at a ratio of 1:100 to 1:1000

Step 5: Prepare 100 ml LB medium with antibiotic
◊ Transfer 100 ml LB medium aseptically to 500-ml conical flask.

◊ Add 200 µl of ampicillin stock solution. Swirl the flask. The final concentration of ampicillin will be 100 µg/ml.

Step 6: Transfer 1 ml starter culture aseptically to 100 ml LB medium.

Step 7: Grow the culture overnight (12 – 16 h) at 37°C with vigorous shaking.
◊ Set the flask in the shaker incubator.

◊ Set the speed 200 – 300 rpm.
◊ Incubate for 12 – 16 h.

Step 8: Take out the culture next morning.

Culture is ready for plasmid isolation. For good plasmid yield, isolate the plasmid on the same day.

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