Quick Protocol for Monarch® DNA Gel Extraction Kit (NEB #T1020)

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Before You Begin:

  • All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).
  • Add 4 volumes of ethanol (≥ 95%) per volume of DNA Wash Buffer. 
  • Please note that the column reservoir holds 800 µl.

Protocol Steps:

  1. Excise the DNA fragment from the agarose gel, taking care to trim excess agarose. Transfer to a 1.5 ml microfuge tube, and weigh the gel slice. Minimize exposure to UV light.
  2. Add 4 volumes of Gel Dissolving Buffer to the gel slice (e.g., 400 μl buffer per 100 mg agarose). If the gel slice is >150 mg, consider reducing the amount of Gel Dissolving Buffer to 3 or 3.5 volumes to minimize the guanidine salt present in the workflow.
  3. Incubate the sample between 37–55°C (typically 50°C), inverting periodically until the gel slice is completely dissolved (generally 5–10 minutes). For DNA fragments > 8 kb, an additional 1.5 volumes of water should be added after the slice is dissolved to mitigate the tighter binding of larger pieces of DNA (e.g., 100 mg gel slice: 400 μl Gel Dissolving Buffer: 150 μl water).
  4. Insert column into collection tube and load sample onto the column. Spin for 1 minute, then discard flow-through.
  5. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
  6. Repeat step 5.
  7. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute.
  8. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, and spin for 1 minute to elute DNA. Typical elution volumes are 6–20 μl. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA (≥ 10 kb), heating the elution buffer to 50°C prior to use can improve yield.
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