Polymerase Chain Reaction (PCR) is a cornerstone technique in molecular biology, enabling the amplification of specific DNA sequences. However, PCR reactions often generate unwanted byproducts or non-specific amplification. Gel extraction is a common method used to purify the desired PCR product from these contaminants.
What is Gel Extraction?
Gel extraction involves separating DNA fragments based on their size using agarose gel electrophoresis. The desired DNA fragment is then excised from the gel and purified.
Why Gel Extraction?
- Removal of Unwanted Byproducts: PCR reactions can generate primer dimers, non-specific amplification products, and other contaminants that can interfere with downstream applications. Gel extraction helps remove these byproducts.
- Isolation of Specific DNA Fragments: Gel extraction allows for the isolation of DNA fragments of a specific size, ensuring that only the desired PCR product is used in subsequent experiments.
- Improved Downstream Results: Purified PCR products are essential for successful cloning, sequencing, and other molecular biology applications. Gel extraction helps improve the quality and reliability of these downstream results.
Gel Extraction Process
- Run PCR Product on Agarose Gel: The PCR product is loaded onto an agarose gel and separated by electrophoresis.
- Visualize DNA with UV Light: The DNA fragments are visualized under UV light and the desired band is excised from the gel.
- Gel Extraction Kit: The excised gel slice is placed in a tube and a gel extraction kit is used to purify the DNA.
- Elution of Purified DNA: The purified DNA is eluted from the spin column and is ready for downstream applications.
Key Points for Successful Gel Extraction
- Use a Sharp Scalpel: When excising the desired DNA band from the gel, use a sharp scalpel to minimize contamination and ensure a clean cut.
- Minimize UV Exposure: UV light can damage DNA. Minimize the exposure time when visualizing the DNA fragments.
- Follow Kit Instructions: Each gel extraction kit has specific instructions. Follow them carefully to ensure optimal results.
Conclusion
Gel extraction is a valuable technique for purifying PCR products, enabling researchers to obtain high-quality DNA for downstream applications. By following the steps outlined above and paying attention to key details, you can successfully purify your PCR products using gel extraction.
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 :
- Add 5 volumes of Dissolving buffer to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene.
- For example, add 500 μl of Dissolving buffer to 100 μl PCR sample .
- Pipet the mixture into the spin column placed in a 2 ml collection tube. Centrifuge at 13,000 rpm for 30-60 s. Discard flow-through and place the column back in the same tube.
- Add 600 µl Washing buffer , and centrifuge at 12,000 rpm for 30 s. Discard the flow-through.
- Repeat above step with another 600 µl Washing 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 (Optional: pre-warm the water to 70–90°C will increase the DNA yield) to the center of the column membrane. Incubate at room temperature for 2-3 min, and centrifuge at 12,000 rpm for 1 min to elute the DNA
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