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. 

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General Guidelines:

Input amount of DNA to be purified should not exceed the binding capacity of the column (5 μg). A starting sample volume of 20–100 μl is recommended. For smaller samples, TE can be used to adjust the volume to the recommended volume range. Centrifugation should be carried out at 16,000 x g in a standard laboratory microcentrifuge at room temperature.

 

Buffer Preparation:

Add ethanol to Monarch DNA Wash Buffer prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch DNA Wash Buffer).

  • For 50-prep kit add 20 ml of ethanol to 5 ml of Monarch DNA Wash Buffer
  • For 250-prep kit add 100 ml of ethanol to 25 ml of Monarch DNA Wash Buffer
Always keep all buffer bottles tightly closed when not in use.

All centrifugation steps should be carried out at 16,000 x g. (~13K RPM in a typical microcentrifuge). This ensures all traces of buffer are eluted at each step.

  1. 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. A starting sample volume of 20–100 μl is recommended. For smaller samples, TE can be used to adjust the volume. 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
    *Please note that recovery of ssDNA < 200 nts can be increased by using the Oligonucleotide Cleanup Protocol, but doing so will shift the cutoff size for DNA binding to 18 nt (versus 50 nt).

  2. Insert column into collection tube and load sample onto column and close the cap. Spin for 1 minute, then discard flow-through.

     To save time, spin for 30 seconds, instead of 1 minute.

     If using a vacuum manifold* instead of centrifugation, insert the column into the manifold and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.

  3. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.

     If using a vacuum manifold, add 200 μl of DNA Wash Buffer and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.

  4. Repeat wash (Step 3).

    * Make sure to follow the manifold manufacturer's instructions to set-up the manifold and connect it properly to a vacuum source.

  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 to ensure traces of salt and ethanol are not carried over to next step.

     If using a vacuum manifold: Since vacuum set-ups can vary, a 1 minute centrifugation is recommended prior to elution to ensure that no traces of salt or ethanol are carried over to the next step.

  6. 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.

    Note: 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.

    Care should be used to ensure the elution buffer is delivered onto the matrix and not the wall of the column to maximize elution efficiency.

     To save time, spin for 30 seconds, instead of 1 minute.