TITLE: Oligonucleotide Cleanup Using Monarch® PCR & DNA Cleanup Kit (5 μg) Protocol (NEB #T1030)
AUTHORS: New England Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The </span><a href="https://www.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Monarch PCR & DNA Cleanup Kit protocol</span></a><span> can be modified to enable the purification of ssDNA, oligonucleotides, and other small DNA fragments. The following modified protocol utilizes the same columns and bind/wash/elute workflow of the Monarch PCR & DNA Cleanup Kit with > 70% recovery and cleanup of oligonucleotides ≥ 15 bp (dsDNA) or ≥18 nt (ssDNA). The Oligonucleotide Cleanup protocol efficiently removes unincorporated nucleotides, short oligos, dyes, enzymes, and salts from labeling and other enzymatic reactions.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/di9f7vcw.jpg" /></div><div class = "text-block"><span>You can also download the Quick Protocol Card </span><a href="https://www.neb.com/-/media/nebus/files/protocols/t1030_quick_protocol_card_monarch_pcrdna_cleanup.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">here</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Oligonucleotide Cleanup]
All centrifugation steps should be carried out at 16,000 xg. (~13K RPM in a typical microcentrifuge). This ensures all traces of buffer are eluted at each step.
?. [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 BufferFor 250-prep kit add 100 ml of ethanol to 25 ml of Monarch DNA Wash BufferAlways keep all buffer bottles tightly closed when not in use.
?. [Oligonucleotide Cleanup]
A starting sample volume of 50 μl is recommended. For smaller samples, nuclease-free water can be used to adjust the volume.
?. [Oligonucleotide Cleanup]
Add  to the 50 μl sample.
[DNA Cleanup Binding Buffer]
?. [Oligonucleotide Cleanup]
Add . Mix well by pipetting up and down or flicking the tube. Do not vortex.
[ethanol (≥ 95%)]
?. [Oligonucleotide Cleanup]
Insert column into collection tube, load sample onto column and close the cap. Spin for  at , 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.* Make sure to follow the manifold manufacturer's instructions to set-up the manifold and connect it properly to a vacuum source.
Centrifuge: 16000 34
?. [Oligonucleotide Cleanup]
Re-insert column into collection tube. Add  and spin for  at . Discard flow-through.If using a vacuum manifold, add 500 μl of DNA Wash Buffer and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.
[DNA Wash Buffer ]
Centrifuge: 16000 34
?. [Oligonucleotide Cleanup]
(Optional): Repeat previous step: Re-insert column into collection tube. Add  and spin for  at . Discard flow-through.If using a vacuum manifold, add 500 μl of DNA Wash Buffer and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.
[DNA Wash Buffer]
Centrifuge: 16000 34
This step is recommended for removal of enzymes that may interfere with downstream applications (e.g., Proteinase K).
?. [Oligonucleotide Cleanup]
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 the 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.
?. [Oligonucleotide Cleanup]
Add ≥  to the center of the matrix. Wait for , then spin for  at  to elute the 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.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.
[DNA Elution Buffer]
Centrifuge: 16000 34