Experimental Procedures for DNA analysis

1. UV Absorbance Method for DNA Quantification

A. Preparation of Blank (Control) Sample

  1. Select a microliter pipette and set its volume to 25 µL.
  2. Aspirate 25 µL of distilled water from the beaker.
  3. Transfer the distilled water into a test sample cuvette.
  4. Again aspirate 25 µL of distilled water.
  5. Transfer it into the blank sample cuvette.
  6. Proceed to measure the blank sample reading.

B. Zeroing (Blank Calibration) of Spectrophotometer

  1. Open the spectrophotometer lid.
  2. Insert the blank cuvette into the spectrophotometer.
  3. Close the lid properly to allow light passage.
  4. Press the Blank button and confirm by pressing Enter.
  5. Measure absorbance at 260 nm (A260).
  6. Measure absorbance at 280 nm (A280).
  7. Proceed to the test sample measurement.

C. Measurement of DNA Sample

  1. Set the microliter pipette to 25 µL.
  2. Aspirate 25 µL of DNA sample from the microcentrifuge tube.
  3. Transfer it into the test sample cuvette.
  4. Open the spectrophotometer lid and insert the DNA sample cuvette.
  5. Close the lid properly.
  6. Press the Test button and confirm by pressing Enter.
  7. Record absorbance at 260 nm (A260).
  8. Record absorbance at 280 nm (A280).
  9. Press A260/A280 to calculate the purity ratio.
  10. Press Conc to calculate the DNA concentration.

2. Agarose Gel Electrophoresis

A. Preparation of Agarose Gel

  1. Weigh 1.5 g of agarose powder using a weighing balance.
  2. Transfer the weighed agarose into a blank flask.
  3. Add 150 mL of TAE/TBE buffer solution to the flask.
  4. Mix the solution thoroughly.

Heating and Cooling of Agarose Solution

  1. Place the flask into a microwave oven.
  2. Heat the mixture for up to 3 minutes until it becomes transparent.
  3. Transfer the hot flask into the cooling system.
  4. Cool the solution at 50°C for 10 minutes.

B. Casting and Solidification of Agarose Gel

  1. Place the casting dam inside the electrophoresis tank.
  2. Insert the comb properly into position.
  3. Set the microliter pipette to 5 µL.
  4. Add 5 µL of ethidium bromide to the agarose solution (handle with safety).
  5. Mix the agarose solution gently.
  6. Pour the mixture into the electrophoresis tank.
  7. Allow the gel to solidify for 20–30 minutes at room temperature.
  8. Remove the casting dam and comb carefully.
  9. Pour TAE/TBE buffer into the tank until the gel is fully submerged.

C. Preparation of DNA Sample

  1. Set the microliter pipette to 100 µL.
  2. Add the loading buffer to the DNA sample.
  3. Mix gently and proceed to loading.

D. Loading of DNA Samples

  1. Set the microliter pipette to 100 µL.
  2. Load the DNA ladder into the first well of the gel.
  3. Load the prepared DNA samples into the remaining wells.

E. Electrophoresis Process

  1. Cover the electrophoresis tank with its protective shield.
  2. Switch on the power supply at 100 V.
  3. Allow electrophoresis to continue until DNA migrates to the desired distance.
  4. Turn off the power supply immediately.

F. Visualization of DNA Bands

  1. Place the gel on the UV transilluminator.
  2. Observe the DNA bands under UV light.
  3. Analyze and document the results using the monitor.