Plasmid DNA Preparation Protocol

BioInnovatise Plasmid DNA Team

Updated December 7, 2023

What Is a Plasmid DNA Preparation Protocol?

A plasmid DNA preparation protocol refers to a systematic set of procedures designed to isolate, purify, and recover plasmid DNA from bacterial, yeast, or mammalian cells. The goal of the protocol is to obtain high-quality DNA that is suitable for various downstream applications, such as molecular cloning, PCR, sequencing, or transfection.

Why Do Plasmid DNA Preparation Protocols Differ?

The choice of plasmid DNA preparation protocol depends on a combination of practical, technical, and application-specific factors. Researchers often select the protocol that best suits their specific needs and resources while ensuring the desired quality and quantity of plasmid DNA for downstream experiments.

Our plasmid prep team uses our own protocol, however, if you have special considerations for your plasmid DNA construct, let us know when submitting your production request. Learn about our quick turnaround plasmid preparation services. The plasmid DNA preparation protocol below is a general protocol for educational purposes.

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The diagram above illustrates the plasmid prep production process for endotoxin free and animal component free plasmid DNA.

Example Plasmid Preparation Protocol for Molecular Biology Applications

Materials Required:

  1. Bacterial culture containing the plasmid of interest
  2. LB broth or appropriate bacterial culture medium
  3. Antibiotics (if required for plasmid selection)
  4. TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
  5. Lysozyme
  6. RNase A (10 mg/ml)
  7. Proteinase K (20 mg/ml)
  8. Sodium dodecyl sulfate (SDS)
  9. Phenol:chloroform:isoamyl alcohol (25:24:1)
  10. Chloroform:isoamyl alcohol (24:1)
  11. Isopropanol
  12. 70% ethanol
  13. RNase-free water

Equipment:

  1. Centrifuge
  2. Microcentrifuge tubes
  3. Water bath or heat block
  4. Vortex mixer
  5. Spectrophotometer

Procedure:

  1. Inoculation of bacterial culture
    1. Start with a single colony of the bacteria containing the plasmid of interest.
    2. Inoculate a suitable volume of LB broth or appropriate bacterial culture medium with the colony.
    3. Add antibiotics if necessary for plasmid selection.
    4. Incubate the culture overnight at 37°C with constant shaking.
  2. Harvesting bacterial cells
    1. Pellet the bacterial cells by centrifugation at 5000 × g for 10 minutes at 4°C.
    2. Discard the supernatant and resuspend the pellet in an equal volume of TE buffer.
  3. Cell lysis
    1. Add lysozyme to a final concentration of 1 mg/ml.
    2. Incubate the mixture at 37°C for 30 minutes.
    3. Add SDS to a final concentration of 0.2% and RNase A to a final concentration of 10 µg/ml.
    4. Incubate at 37°C for an additional 30 minutes.
  4. Protein digestion
    1. Add Proteinase K to a final concentration of 20 µg/ml.
    2. Incubate at 55°C for 1–2 hours or until the solution becomes clear.
  5. Extraction of plasmid DNA
    1. Add an equal volume of phenol:chloroform:isoamyl alcohol.
    2. Vortex the mixture thoroughly and centrifuge at 10,000 × g for 10 minutes.
    3. Carefully transfer the aqueous phase to a new tube.
    4. Repeat the extraction with chloroform:isoamyl alcohol.
    5. Precipitate the plasmid DNA by adding 0.6 volumes of isopropanol.
    6. Centrifuge at 10,000 × g for 15 minutes.
  6. Washing and elution
    1. Wash the DNA pellet with 70% ethanol and air dry.
    2. Resuspend the pellet in an appropriate volume of RNase-free water.
  7. Quality assessment
    1. Measure the concentration and purity of the isolated plasmid DNA using a spectrophotometer.
    2. Optionally, run an agarose gel to check the integrity of the DNA.
  8. Storage
    1. Store the purified plasmid DNA at -20°C for short-term storage or -80°C for long-term storage.

Note: All steps should be performed using sterile techniques to avoid contamination. Adjust volumes and concentrations according to the scale of the culture and the plasmid copy number.

What Is the Difference Between Mini Prep, Midi Prep, and Maxi Prep?

Plasmid mini prep, midi prep, and maxi prep are different methods for isolating plasmid DNA from bacterial cultures, and they vary in scale, yield, and the amount of starting bacterial culture. Each method is suited for specific applications based on the amount of plasmid DNA required.

See the breakdown of mini prep, midi prep, and maxi prep by scale, starting culture volume, yield, applications, method, and deliverable volume our plasmid DNA preparation services.

Want to learn more about the latest in plasmid research? Our colleagues at ScienceDirect and the American Society for Biochemistry & Molecular Biology continuously collect and publish the latest information and research.

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