Simple Autoclave Diagram

Isolation of Plasmid DNA from Escherichia coli by Alkaline Lysis Method

Aim: To isolate plasmid DNA from Escherichia coli using the alkaline lysis method and confirm its presence by agarose gel electrophoresis.

Principle: Plasmid DNA is small, circular and separate from chromosomal DNA. That is why we can isolate it selectively. In alkaline lysis, cells are first broken open using SDS and NaOH. SDS disrupts membrane and proteins. NaOH denatures DNA. Now comes the interesting part. When we neutralize with potassium acetate, chromosomal DNA and proteins precipitate out because they are large and tangled. Plasmid DNA, being small and circular, renatures properly and stays in solution. After centrifugation, supernatant contains plasmid DNA. Then we precipitate it using alcohol. Simple idea, but timing matters.

Requirements:

• Overnight culture of Escherichia coli in LB broth
• LB broth: Tryptone 10 g/L, Yeast extract 5 g/L, NaCl 10 g/L
• Solution I (Resuspension buffer): Glucose 50 g/L, Tris-HCl 25 mM, EDTA 10 mM
• Solution II (Lysis buffer): NaOH 0.2 N, SDS 10 g/L
• Solution III (Neutralization buffer): Potassium acetate 5 M, acetic acid adjusted to pH 5.5
• Isopropanol
• 70% Ethanol
• TE buffer: Tris-HCl 10 mM, EDTA 1 mM
• Microcentrifuge tubes
• Micropipettes and tips
• Centrifuge
• Water bath or incubator
• Ice box

For Agarose Gel Electrophoresis:

• Agarose powder (0.8% gel: 8 g/L in 1X TAE buffer)
• 50X TAE buffer stock: Tris base 242 g/L, Glacial acetic acid 57.1 mL/L, EDTA 0.5 M solution 100 mL/L, adjust to pH 8.0
• Ethidium bromide solution (10 mg/mL, handle with gloves, it is a mutagen) or any safe alternative like GelRed
• 6X DNA loading dye: Bromophenol blue 0.25% w/v, Xylene cyanol 0.25% w/v, Glycerol 30% v/v in water
• DNA ladder (100 bp or 1 kb ladder, whichever is available)

Procedure:

Day 1: Starting the culture

• Take a single colony of E. coli carrying the plasmid from an LB agar plate (ampicillin plate) and inoculate into 5 mL of LB broth containing ampicillin (50 microgram/mL final concentration).
• Incubate the culture overnight at 37 °C with constant shaking at 200 rpm on an orbital shaker. The culture should appear turbid next morning, this confirms good growth.

Day 2: Plasmid isolation

• Transfer 1.5 mL of the overnight culture into a labelled 1.5 mL microcentrifuge tube. Centrifuge at 8000 rpm for 2 minutes at room temperature. Discard the supernatant by pipetting, not by pouring, be careful not to disturb the pellet.
• Repeat step 3 to get a denser cell pellet if needed. Completely remove the residual liquid by inverting the tube briefly on tissue paper.
• Resuspend the cell pellet in 100 microlitre of ice-cold Solution I. Add 2 microlitre of RNase A (10 mg/mL) at this step itself. Pipette up and down gently until the pellet is fully homogeneous and no clumps remain.
• Add 200 microlitre of freshly prepared Solution II. Mix by gently inverting the tube 6 to 8 times. Do not vortex. The solution will become slightly viscous and clear. This means lysis is happening. Incubate at room temperature for not more than 5 minutes.
• Add 150 microlitre of ice-cold Solution III. Again, mix by inverting the tube 10 to 12 times. You will see white precipitate forming immediately. This is the chromosomal DNA, SDS, and cell proteins coming out of solution.
• Centrifuge immediately at 12000 rpm for 10 minutes at 4 degrees C. A compact white pellet will form at the bottom. Your plasmid is in the clear supernatant above.
• Carefully transfer the supernatant into a fresh 1.5 mL tube without disturbing the pellet. If the supernatant looks slightly cloudy, centrifuge once more for 5 minutes.
• Add 0.6 volumes of chilled isopropanol to the collected supernatant (roughly 270 microlitre if you collected 450 microlitre). Mix gently by inversion. Keep the tube at -20 degrees C for 20 to 30 minutes. DNA will precipitate.
• Centrifuge at 12000 rpm for 15 minutes at 4 degrees C. Discard the supernatant carefully, the pellet may not always be visible at this point, it is there. Do not panic.
• Wash the pellet with 500 microlitre of 70% ethanol. Centrifuge at 10000 rpm for 5 minutes. Discard the ethanol wash carefully.
• Air dry the pellet for 10 to 15 minutes at room temperature. Do not over-dry. Once the pellet looks translucent (or even invisible), it is ready.
• Dissolve the pellet in 30 to 50 microlitre of TE buffer. Store at -20 degrees C if not using immediately.

Day 2 or Day 3: Agarose gel electrophoresis

• Prepare 0.8% agarose gel in 1X TAE buffer. Add ethidium bromide to a final concentration of 0.5 microgram/mL after the agarose cools to around 55 degrees C (or add GelRed as per supplier instructions). Pour into gel tray, insert comb, and allow it to solidify for 30 minutes.\
• Mix 5 microlitre of isolated plasmid DNA with 1 microlitre of 6X loading dye. Load this into a well. Load the DNA ladder in a separate well.
• Run electrophoresis at 80 to 100 V in 1X TAE buffer for 45 minutes to 1 hour.
• Visualize the gel under UV transilluminator or gel documentation system. Note and photograph the bands observed.

Observation: On the agarose gel, two or three distinct bands are typically visible for the isolated plasmid: the supercoiled form migrates fastest, the nicked circular (relaxed) form runs slowest, and the linear form (if present) migrates between the two. No prominent smear should be visible, which would indicate RNA or chromosomal DNA contamination.

Result: Plasmid DNA was successfully isolated from E. coli and confirmed by the presence of characteristic supercoiled and relaxed circular bands on 0.8% agarose gel under UV illumination after ethidium bromide staining.

Conclusion: This method works well for small scale plasmid isolation in basic lab conditions. I have seen students mess up mainly during mixing step, too rough handling shears chromosomal DNA and contaminates the sample. If done carefully, you get clean plasmid DNA which can be used for further work like restriction digestion or PCR.

Note: This protocol is for educational use in standard teaching laboratories. Small variations may occur depending on lab conditions and handling. Results may vary. biologynotes.in cannot take responsibility for any errors, misuse, or results obtained. Always follow proper lab safety and instructor guidance.