Essential Tips for Successful DNA Gel Extraction

Extracting DNA from agarose gels is a crucial step in many molecular biology workflows. Agarose gel electrophoresis is an effective and precise way to separate DNA fragments by size, allowing selective extraction of specific fragments needed for downstream experiments. The following steps are part of the Monarch Spin DNA Gel Extraction Kit (NEB #T1120) workflow, but they are general best practices that can be applied to most gel extraction protocols. A gel extraction kit offers a streamlined process, ensuring high recovery and purity of DNA by eliminating buffer retention and carryover contamination. Here are some pointers to help you get the most out of your gel extractions.

 

(1) DISSOLVE YOUR GEL SLICE COMPLETELY

Dissolving the gel completely is essential for high DNA recovery, as incomplete dissolution can lead to clogging of the column. Agarose gels using standard laboratory grade or low-melt agarose are compatible up to concentrations of 4%. Tris-Acetate-EDTA (TAE) buffer is a commonly used running buffer, especially if you’re planning to use the DNA for downstream experiments; however, Tris-Borate-EDTA (TBE) buffer can also be used.

 

• Use a clean, sharp blade to make precise cuts in the gel.
• Trim the excess gel as much as possible! Minimizing the gel surrounding your band will reduce the volume of binding buffer required and increase your DNA yield.
• Minimize the duration of DNA exposure to UV light, as prolonged exposure can damage the DNA.
• Ensure the gel is fully submerged in the buffer and incubate at 50°C for 5–10 minutes. Vortexing the tube occasionally can help accelerate the melting process.
• Extend the incubation time if a lower temperature and/or higher concentration of agarose gel is used (>2%) to ensure complete dissolving of the gel.
• Extend the incubation time for particularly large or thick slices to ensure complete dissolution.

 

• Don’t store the gel slice for long periods before extraction. The gel slice can be stored in a closed microfuge tube at 4°C for up to 3 days; however, the best results will be achieved if you extract your band immediately following excision.

 

 

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(2) BIND YOUR DNA EFFICIENTLY

To improve the binding step, closely monitor your column capacity, centrifugation time, and temperature.

 

• Let the solution cool to room temperature before proceeding.
• After loading the dissolved gel and buffer mixture on the column, spin it at 16,000 x g for a full minute to ensure the DNA binds effectively to the column matrix.

 

• Don’t overload the column by adding more than 800 µl or >5 µg. If using the Monarch Spin DNA Gel Extraction Kit (#T1120), the maximum binding capacity of the column is 5 μg of purified DNA. The size recovery of DNA ranges from 50 bp to 25 kb.

 

 

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(3) WASH YOUR DNA THOROUGHLY

Thorough washing is key to achieving high-purity DNA.

 

• Use the provided wash buffer to rinse the column twice to ensure efficient removal of any residual salt carry-over.
• Follow each wash with a full-speed spin to remove any residual contaminants.
• After the final wash, spin the column for an additional minute to remove all traces of ethanol. Additional centrifugation is unnecessary if using Monarch Spin DNA Gel Extraction Kit (#T1120).
  Note: The wash buffer, Monarch Buffer WZ, is an ethanol-based buffer designed for optimal DNA purity.

 

• Don’t let the tip of the column touch the flow-through. If in doubt, spin again to remove all residual wash buffer.

 

 

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(4) ELUTE CAREFULLY

Elution is a critical step in the recovery of your DNA. The optimal range for elution volume is 5-20 μl of elution buffer.

 

• Use the elution buffer in the kit and pre-warm it to 50°C to increase yield.
• Ensure the column is free of residual wash buffers before adding the elution buffer.
• Apply the buffer directly to the center of the column matrix.     
• For long-term storage of the DNA, we recommend using the supplied elution buffer.
  Note: The Monarch Spin DNA Gel Extraction Kit contains the Monarch Buffer EY (10 mM Tris, 0.1 mM EDTA, pH 8.5).
• Use modified elution methods to increase the recovery of longer DNA, such as a heated elution buffer (50°C) or incubating at room temperature for 5 minutes after adding elution buffer. Longer DNA fragments bind tighter to the matrix, which may result in inefficient elution without these modifications.

 

• Don’t shorten or skip the incubation - incubate for a full minute.
• Don’t store the sample for an extended amount of time if you have eluted it in water rather than an elution buffer.
  Note: If eluting in water, for maximum elution efficiency, ensure the water is nuclease-free and has a pH between 7 to 8.5. Milli-Q™ water is often slightly acidic, requiring pH adjustment before it can be used for elution.

 

 

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(5) MONITOR YOUR YIELD AND PURITY

After elution, you can use a spectrophotometer to measure the concentration and purity of your DNA. This ensures that your sample is ready for downstream applications without any contaminants that could interfere with your experiments.

Carried-over salts will be indicated by a low A_260/230 ratio, which is why the column tip must not touch the flow through.

If you observe a faint additional band running below the expected size on a gel, it may be due to DNA denaturation. Chaotropic agents used in silica-based DNA purification can induce DNA denaturation, causing single-stranded forms of DNA to have faster mobility in a gel. To renature your sample, add NaCl to 10 mM and heat the sample to 95°C for one minute, then slowly cool to room temperature.

 

 

 

By following these tips, you can achieve consistent, high-quality DNA recovery from agarose gels, which will support the success of your projects. Happy extracting!

 

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