Chapter 4 : DNA Extraction From Agarose Gel

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Agarose gel electrophoresis is a cornerstone technique in molecular biology, used to separate DNA fragments based on size. However, once the DNA is nestled within the gel, how do you extract it for further analysis or manipulation? This blog post will outline a simple and effective method for DNA extraction from agarose gel.

Why Extract DNA from Agarose Gel?

Extracting DNA from agarose gel opens doors to a multitude of downstream applications, including:

  • Cloning: Inserting the isolated DNA fragment into a vector for further study or expression.
  • Sequencing: Determining the precise nucleotide sequence of the DNA fragment.
  • Restriction Enzyme Digestion: Cutting the DNA fragment at specific recognition sites for analysis or manipulation.
  • PCR Amplification: Making millions of copies of the DNA fragment for further analysis or experimentation.

Materials Needed

  • Agarose gel containing the DNA fragment of interest
  • Clean scalpel or razor blade
  • Microcentrifuge tube
  • DNA extraction kit or buffer
  • Microcentrifuge

Step-by-Step Procedure

  1. Gel Excision:
    • Visualize the DNA fragment within the agarose gel using a UV transilluminator.
    • Carefully excise the band containing the DNA fragment using a clean scalpel or razor blade. Minimize the amount of excess gel to avoid impurities.
  2. Gel Dissolution:
  1. Place the excised gel slice into a microcentrifuge tube.
  2. Add an appropriate amount of DNA extraction buffer or kit reagents to the tube.
  3. Follow the manufacturer’s instructions for dissolving the gel and releasing the DNA. This often involves incubation at an elevated temperature or the addition of chaotropic salts.
  4. DNA Purification:
  5. The DNA extraction kit or buffer will often include a purification step to remove impurities such as salts, proteins, and residual agarose. This may involve binding the DNA to a silica membrane or using a spin column.
  6. DNA Elution:
  7. After purification, the DNA is eluted from the membrane or column using a small volume of elution buffer or water.
  8. DNA Quantification and Analysis:
  1. The concentration and purity of the extracted DNA can be assessed using spectrophotometry or fluorometry.
  2. The DNA is now ready for downstream applications such as cloning, sequencing, or PCR.

Tips for Success

  • Use a clean scalpel or razor blade to minimize contamination.
  • Minimize the amount of excess gel excised to avoid impurities.
  • Follow the manufacturer’s instructions for the DNA extraction kit or buffer carefully.
  • Avoid excessive exposure of the DNA to UV light during visualisation.

Conclusion

DNA extraction from agarose gel is a fundamental technique in molecular biology that enables further analysis and manipulation of DNA fragments. By following this simple guide and using a reliable DNA extraction kit or buffer, you can successfully isolate DNA from agarose gel for your research needs.

Dissolving Buffer/Extraction buffer :

Ingredients for gel dissolving buffers Guanidine thiocyanate (GuSCN), Tris-Cl solution, Dilute hydrochloric acid (HCl), and Sterile water. 

Washing buffer :

DNA Wash Buffer (80% ethanol, 20 mM NaCl, 2 mM Tris-HCl, pH 7.5)

Elution buffer :

(0.5 mM EDTA, 10 mM Tris-HCl, pH 9.0 )


Protocol :

  • Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
    Note: Minimize gel volume by visualising DNA and cutting the smallest possible gel slice.
  • Place excised agarose gel slice in a sterile 1.5 ml microcentrifuge tube. Determine gel mass by first pre-weighing the tube, and then re-weighing the tube with the excised gel slice.
  • Add 3 volumes of Dissolving Buffer  to 1 volume of gel (100 mg~100 μl). For example, add 300 μl of Dissolving Buffer to each 100 mg of gel.
  • Incubate at 56°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
    IMPORTANT: Solubilise agarose completely. For >2% gels, increase incubation time.
  • After the agarose gel slice is completely dissolved, add 150μl isopropanol for every 100 mg
    agarose gel slice to the tube. Vortex thoroughly.
    For example, if the agarose gel slice is 100 mg, add 150 μl isopropanol. Do not centrifuge the sample at this stage.
  • Pipet the mixture into the spin column placed in a 2 ml collection tube . Centrifuge at 13,000 rpm for 60 s. Discard flow-through and place the column back in the same tube.
    The maximum volume of the column reservoir is 720 μl. For sample volumes of more than 720 μl, simply load and spin again.
  • Add 600 μl Wash Buffer , and centrifuge at 12,000 rpm for 30 s. Discard the flow-through.
  • Repeat above step with another 600 μl Buffer Wash buffer.
  • Place the spin column back into the same collection tube. Centrifuge the empty column at 13,000 rpm for 2 min to completely remove ethanol from the column. 
  • Note: It is important to dry the membrane of the spin column, since residual ethanol may interfere with subsequent reactions. This centrifugation step ensures that no residual ethanol will be carried over during the following elution. 
  • Place the column in a clean 1.5 ml microcentrifuge tube. Add 50 μl of Elution Buffer .

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