Plasmid DNA Preparation Protocol

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.