Quick Protocol for Oligonucleotide Cleanup Using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030)
DNA Cleanup and Concentration: for the purification of up to 5 μg of DNA (ssDNA > 200 nt and dsDNA > 50 bp) from PCR and other enzymatic reactions. A detailed protocol and a quick protocol are available for your convenience.
Oligonucleotide Cleanup (below): for the purification of up to 5 μg of DNA fragments ≥ 15 bp (dsDNA) or ≥ 18 nt (ssDNA). Expected recovery is > 70%. When purifying ssDNA of any size, recovery can be increased by using this protocol; however, it is important to note that this protocol shifts the cutoff for smaller fragments to 18 nt (rather than 50 nt for the DNA Cleanup and Concentration Protocol).
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.
Oligonucleotide Cleanup Protocol Steps:
Add 100 μl DNA Cleanup Binding Buffer to the 50 μl sample. We recommend a sample volume of 50 μl. For smaller samples, adjust the volume with nuclease-free water.
- Add 300 μl ethanol (≥ 95%). Mix well by pipetting up and down or flicking the tube. Do not vortex.
- Insert column into collection tube and load sample onto column. Spin for 1 minute, then discard flow-through.
- Re-insert column into collection tube. Add 500 μl DNA Wash Buffer and spin for 1 minute. Discard flow-through.
- Repeat step 4 (Optional). Recommended for removal of enzymes that may interfere with downstream applications (e.g., Proteinase K).
- 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, then 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.