The Isolation of plasmid DNA is explained in this section
Aim of the experiment: To isolate plasmid DNA from bacterial cells.
Principle of plasmid DNA Isolation
When bacteria are lysed under alkaline conditions both DNA and proteins are
precipitated. After the addition of acetate-containing neutralization buffer the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids can renature and stay in solution.
In prokaryotes, plasmid is double stranded, circular, and is found in the cytoplasm. The cell membranes must be disrupted in order to release the plasmid in the extraction buffer. Solution 1 contains glucose, Tris, and EDTA. Glucose provides osmotic shock leading to the disruption of cell membrane, Tris is a buffering agent used to maintain a constant pH8. Plasmid can be protected from endogenous nucleases by chelating Mg2++ ions using EDTA. Mg2++ ion is considered as a necessary cofactor for most nucleases. Solution II contains NaOH and SDS and this alkaline solution is used to disrupt the cell membrane and NaOH also denatures the DNA into single strands. Solution III contains acetic acid to neutralise the pH and potassium acetate to precipitate the chromosomal DNA, proteins, along with the cellular debris. Phenol /chloroform is used to denature and separate proteins from plasmid. Chloroform is also a protein denaturant, which stabilizes the rather unstable boundary between an aqueous phase and pure phenol layer. The denatured proteins form a layer at the interface between the aqueous and the organic phases which are removed by centrifugation. Once the plasmid DNA is released, it must be precipitated in alcohol. The
plasmid DNA in the aqueous phase is precipitated with cold (0oC) ethanol or isopropanol. The precipitate is usually redissolved in buffer and treated with phenol or organic solvent to remove the last traces of protein, followed by reprecipitation with cold ethanol.
Illustration of plasmid DNA isolation
MATERIALS REQUIRED FOR PLASMID DNA ISOLATION
- Luria Broth
- Bacterial cells containing plasmid
- Tris EDTA (TE) buffer(pH 8.0)
- Solution I
- Solution II
- Solution III
- Phenol-chloroform mixture
- 70% ethanol
- Autoclaved Distilled Water
- Eppendorf tubes 2ml
PREPARATION OF REAGENTS REQUIRED FOR PLASMID DNA ISOLATION
- TE BUFFER (pH 8.0): 10 mm Tris HCl (pH 8.0) 1 mm EDTA (pH 8.0)
2. Solution I: Lysis solution
3. Solution II: Denaturing solution
3. Solution III: Neutralizing solution
4. PHENOL – CHLOROFORM MIXTURE: Mix equal volume of phenol
with chloroform. Keep the mixture on ice and add 20 ml TE buffer, extract by shaking for 15 minutes. Remove the dust on the surface layer using a pipette. Repeat 4-5 times. Add 30-40 ml of TE buffer and store it in dark.
PROCEDURE FOR PLASMID DNA ISOLATION
- Take 2 ml overnight culture and harvest cells by centrifugation for 5 minutes. Discard the supernatant carefully.
- Add 100 µl of solution I to the cell pellet and resuspend the cells by gentle mixing.
- Incubate the above mixture at room temperature for 5 minutes.
- Add 200 µl of solution II to the mixture and mix by inverting the tubes for 5 minutes.
- Incubate 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 at 10,000 rpm for 5 minutes.
- Transfer the supernatant into fresh tube.
- Add 400 µl of phenol-chloroform mixture to the contents, mix well by inverting and incubate them at room temperature for 5 minutes.
- Centrifuge at 10000 rpm for 5 minutes.
- Collect the supernatant (viscous) using cut tips and transfer to a fresh tube.
- Add 0.8 ml of isopropanol and mix gently by inversion. Incubate for 30 min at room temperature.
- Centrifuge the contents at 10,000 rpm for 10 minutes.
- Discard the supernatant after centrifugation.
- After air drying for 5 minutes, add 100 µl of TE buffer or autoclaved distilled water to the pellet to resuspend the plasmid DNA. The contaminated salt in the DNA pellet can be removed with 70% ethanol washing.
- Take 10 µl of plasmid sample and dilute to 1 ml with distilled water for spectrometric analysis.
- The concentration of plasmid is determined using a spectrophotometer at 260/280 nm.
- An aliquot of plasmid DNA is used for agarose electrophoresis for quantitative and qualitative analyses.
- Cut tips should be used so that the plasmid is not subjected to mechanical disruption.
- The phenol chloroform extraction should be repeated depending on the source of plasmid to obtain pure plasmid.
- DNase free plastic wares and reagents should be used.
Results and Discussion:
References: DEPARTMENT OF BIOTECHNOLOGY, SCHOOL OF BIOENGINEERING, SRM UNIVERSITY