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%) to one volume of DNA Wash Buffer.
- Please note: column holds 800 µl.
- 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.
- Add 4 volumes of Gel Dissolving Buffer to the gel slice (e.g., 400 μl buffer per 100 μl or 100 mg agarose).
- Incubate the sample between 37–55°C (typically 50°C), vortexing 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 μl gel slice: 400 μl Gel Dissolving Buffer: 150 μl water).
- Insert column into collection tube and load sample onto the column. Spin for 1 minute, then discard flow-through.
- Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
- Repeat step 5.
- 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.
- 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.