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Using the NEB #T1020 Monarch Kit
For a detailed protocol, or to download the full manual, visit www.neb.com/T1020
BEFORE YOU BEGIN:
- Add 4 volumes of ethanol (2 95%) to one volume of DNA Wash Buffer. (Needed to do the first time that the kit is used).
- All centrifugation steps should be carried out at 16,000 xg (-13,000 RPM).
- Please note: column holds 800 ul.
- Run your digested DNA on an agarose gel. Use the 'wide combs' that will allow for more volume inside of them. Run the gel slowly (40-60 volts) for better band separation.
- Weigh empty micro-centrifuge tubes, and write the weight on the outside of the tube.
- Using the UV illuminator in Exx (across from Superlab), excise the DNA fragment from the agarose gel, taking care to trim excess agarose. Transfer to the 1.5 ml micro-centrifuge tube. Minimize exposure to UV light. Weigh the gel slice + micro-centrifuge tube and calculate the weight of just the agarose chunk. Agarose in a tube can be frozen at -20 and extracted later — this is a potential stopping point in the protocol.
- Turn on the small water bath and set to 50°C. Put in the DNA Elution Buffer bottle, ensuring that it does not tip over in the water bath.
- Add 4 volumes of Gel Dissolving Buffer to the gel slice (e.g., 400 ul buffer per 100 ul or 100 mg agarose).
- Incubate the sample at 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 ul gel slice: 400 ul Gel Dissolving Buffer: 150ul water).
- Insert column into collection tube and load sample onto the column with the mixture. Remember that the column can hold only 800ul maximum; you may need to load 800ul; spin; discard flow-through; load remaining liquid; spin again. Spin for 1 minute, then discard flow-through.
- Re-insert column into collection tube. Add 200 ul DNA Wash Buffer and spin for 1 minute. Discarding flow-through.
- Repeat step 5 by adding 200 ul DNA Wash Buffer and spin for 1 minute.
- Transfer column to a clean 1.5 ml micro-centrifuge 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 6ul - 20ul of DNA Elution Buffer at 50°C to the center of the matrix. Wait for 1 minute, and spin for 1 minute to elute DNA. Molecular grade 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.