Quick Protocol for DNA Cleanup and Concentration Using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030)
There are two protocols available for this product:
DNA Cleanup and Concentration (below): for the purification of up to 5 μg of DNA (ssDNA > 200 nt and dsDNA > 50 bp) from PCR and other enzymatic reactions.
Oligonucleotide Cleanup: 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). A detailed protocol and quick protocol are available for your convenience.
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.
DNA Cleanup and Concentration Protocol Steps:
- Dilute sample with DNA Cleanup Binding Buffer according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex. We recommend a sample volume of 20–100 μl. For smaller samples, adjust the volume with TE. For diluted samples larger than 800 μl, load a portion of the sample, proceed with step 2, and then repeat as necessary.
Sample Type Ratio of Binding Buffer: Sample Example dsDNA > 2 kb (plasmids, gDNA) 2:1 200 μl: 100 μl dsDNA < 2 kb (some amplicons, fragments) 5:1 500 μl: 100 μl ssDNA > 200 nt* 7:1 700 μl: 100 μl
- 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 200 μl DNA Wash Buffer and spin
for 1 minute. Discarding flow-through is optional.
- Repeat step 3.
- 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. For larger size DNA (≥ 10 kb), heating the elution buffer
to 50°C prior to use can improve yield.