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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol for DNA Cleanup and Concentration Using the Monarch® PCR & DNA Cleanup Kit (5 μg) (</span><a href="https://www.neb.com/products/t1030-monarch-pcr-dna-cleanup-kit-5-ug#Product%20Information" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">NEB #T1030</span></a><span>).</span></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]
?. [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.All centrifugation steps should be carried out at 16,000 xg (~13K RPM in a typical microcentrifuge).
?. [DNA Cleanup and Concentration]
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 the next step, and then repeat as necessary.SAMPLE TYPERATIO OF BINDING BUFFER:SAMPLEEXAMPLEdsDNA > 2 kb (plasmids, gDNA)2:1200 μl:100 μldsDNA < 2 kb
(some amplicons, fragments)5:1500 μl:100 μlssDNA > 200 nt*7:1700 μl:100 μl
SAMPLE TYPERATIO OF BINDING BUFFER:SAMPLEEXAMPLEdsDNA > 2 kb (plasmids, gDNA)2:1200 μl:100 μldsDNA < 2 kb
(some amplicons, fragments)5:1500 μl:100 μlssDNA > 200 nt*7:1700 μl:100 μl
?. [DNA Cleanup and Concentration]
Insert column into collection tube and 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
?. [DNA Cleanup and Concentration]
Re-insert column into collection tube. Add  and spin for  at . 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.
[DNA Wash Buffer]
Centrifuge: 16000 34
?. [DNA Cleanup and Concentration]
Repeat wash: Re-insert column into collection tube. Add  and spin for  at . 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.
[DNA Wash Buffer]
Centrifuge: 16000 34
?. [DNA Cleanup and Concentration]
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.
?. [DNA Cleanup and Concentration]
Add ≥  to the center of the matrix. Wait for , then spin for  at  to elute DNA.To save time, spin for 30 seconds, instead of 1 minute.
[DNA Elution Buffer]
Centrifuge: 16000 34
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.