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.

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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. 

DNA Cleanup and Concentration Protocol Steps:

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. 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

   *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. Spin for 1 minute, then discard flow-through.
   
   
3. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
   
   
4. Repeat step 3.
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.
   
   
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. 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.