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Plasmid DNA Isolation from Bacteria


In bacteria, the Plasmid is present in the cytoplasm. To get the plasmid DNA out of the bacteria, its cell membranes must be disrupted in order to get the Plasmid DNA in the extraction buffer. There are three main solutions involves in the procedure. 

Solution I: It contains Glucose, Tris and EDTA.. Glucose provides the Osmotic shock which leads to the disruption of the cell membrane. Tris  is buffering agent and maintains constant pH 8. EDTA is used to protect the Plasmid from endogenous nucleases by chelating the magnesium ions. 

Solution II: it contains NaOH and SDS. This is an Alkaline solution. It is used to disrupt the cell and NaOH also denatures the DNA into single strands. 

Solution III: it contains the acetic acid and potassium acetate. Acetic acid is used to neutralize the pH. and Potassium acetate is used to precipitate the chromosomal DNA, proteins and other cellular debris. 

Phenol/Chloroform: It is used to denature and separate proteins from Plasmid. Chloroform stabilizes the boundary between an aqueous phase and pure phenol layer. Denatured proteins form a layer at the interface between aqueous and organic phases, which are removed by the centrifugation. 

(Once the plasmid is released it must be precipitated with alcohol such as cold ethanol or isopropanol). 

Procedure:
  • Take 2mL of overnight culture and centrifuge for 5 minutes. Discard the supernatant carefully. Do not disturb the pellet.
  • Add 100µL of Solution I to the pellet and re suspend the cells using pipette.
  • Incubate the mixture for 5 minutes at room temperature.
  • Add 200µL of Solution II to the mixture and mix by inverting the tubes for 5 minutes.
  • Incubate the mixture for 5-10 minutes at room temperature.
  • Add 500µL of ice cold Solution III to the mixture and mix by inverting the tube.
  • Incubate on ice for 10 minutes.
  • Centrifuge the tubes at 10,000 rpm for 5 minutes.
  • Transfer the supernatant into a fresh tube.
  • Add 400µL of phenol-chloroform mixture to the contents and mix well by inverting the tube for 3-4 times and incubate at room temperature for 5 minutes.
  • Centrifuge the tubes at 10000 rpm for 5 minutes.
  • Collect the viscous supernatant using cut tips and and transfer it to a fresh tube.
  • Add 800µL of isopropanol and mix gently by inversion. Incubate it for 30 minutes at room temperature.
  • Centrifuge at 10,000 rpm for 10 minutes.
  • Discard the supernatant.. Do not disturb the pellet.
  • Keep the tubes open for air drying for 5-10 minutes.
  • After air drying, add 100µL of TE buffer or distilled water and resuspend. (The contaminated salt in the DNA can be removed with 70% ethanol washing). 
Plasmid DNA Isolation
Image Source: srmuniv

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