DNA CLEANUP AND CONCENTRATION
PROTOCOL STEPS:
1.	 Dilute sample with DNA Cleanup Binding Buffer (ensure that
isopropanol has been added, as indicated on the bottle label)
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 repeat as necessary.
BEFORE YOU BEGIN:
•	IMPORTANT UPDATE: Add 0.36 volumes of isopropanol to one
volume of DNA Cleanup Binding Buffer (e.g., 63.5 ml isopropanol
to 175 ml buffer)
•	Add 4 volumes of ≥ 95% ethanol to one volume of DNA Wash
Buffer (e.g., 100 ml ethanol to 25 ml buffer)
•	All centrifugation steps should be carried out at
16,000 x g (~13,000 RPM)
•	If working with DNA fragments ≥ 10 kb, preheat the
appropriate amount of DNA Elution Buffer to 50°C
THERE ARE TWO PROTOCOLS AVAILABLE
FOR THIS PRODUCT:
•	DNA Cleanup and Concentration: 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).
NEB #T1030
continued on back →
For a detailed protocol or to download the
full manual, visit www.neb.com/ T1030.
Monarch
®
PCR & DNA Cleanup Kit (5 μg) Protocol Card
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.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information about commercial rights,
please email us at busdev@neb.com. The use of this product may require you to obtain additional third party intellectual property rights for certain applications.
New England Biolabs is an ISO 9001, ISO 14001 and ISO 13485 certified facility.
© Copyright 2021, New England Biolabs, Inc.; all rights reserved.
This card is made with FSC certified 100% post-consumer fiber. Please recycle.
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.
OLIGONUCLEOTIDE CLEANUP PROTOCOL STEPS:
1.	 Add 100 μl DNA Cleanup Binding Buffer (ensure that
isopropanol has been added, as indicated on the bottle label)
to the 50 μl sample. We recommend a sample volume of 50 μl.
For smaller samples, adjust the volume with nuclease-free water.
2.	 Add 300 μl ethanol (≥ 95%). Mix well by pipetting up and down
or flicking the tube. Do not vortex.
3.	 Insert column into collection tube and load sample onto column.
Spin for 1 minute, then discard flow-through.
4.	 Re-insert column into collection tube. Add 500 μl DNA Wash
Buffer and spin for 1 minute. Discard flow-through.
5.	 (Optional) Repeat step 4. This second wash step is optional,
but recommended for removal of enzymes that may interfere with
downstream applications (e.g., Proteinase K). Please note that if
carrying out a second wash step, additional DNA Wash Buffer may
be required.
6.	 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.
7.	 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.
Want to use this kit to purify DNA from agarose gels?
Simply purchase the Monarch Gel Dissolving Buffer
(NEB #T1021L) and use with this kit. Protocol available
at www.neb.com/T1020
Questions?
Our tech support scientists would be happy to help.
Email us at info@neb.com
V4.1 – 05.21 #101031