Quick Protocol for DNA Cleanup and Concentration Using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) V.6
- New England Biolabs1
- 1New England Biolabs
External link: https://www.neb.com/protocols/2015/12/08/quick-protocol-for-monarch-pcr-dna-cleanup-kit-5-g-t1030
Protocol Citation: New England Biolabs . Quick Protocol for DNA Cleanup and Concentration Using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030). protocols.io https://dx.doi.org/10.17504/protocols.io.bg9rjz56Version created by Isabel Gautreau
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: June 07, 2020
Last Modified: February 25, 2022
Protocol Integer ID: 37905
Keywords: Monarch PCR Cleanup Kit, Monarch DNA cleanup kit
Abstract
This is the "quick" version of Monarch® PCR & DNA Cleanup Kit (5 μg) Protocol (NEB #T1030) DNA Cleanup and Concentration. For the full protocol, please click here.
Guidelines
For detailed protocol and more information, visit www.neb.com/T1030
The full protocol is available here.
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.
Materials
MATERIALS
Monarch® PCR & DNA Cleanup Kit (5 μg)New England BiolabsCatalog #T1030
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Add isopropanol to Monarch DNA Cleanup Binding Buffer prior to use*:
- For the 50-prep kit, add 14 mL of isopropanol to the DNA Cleanup Binding Buffer.
- For the 250-prep kit, add 63.5 mL of isopropanol to the DNA Cleanup Binding Buffer.
Add ethanol to Monarch DNA Wash Buffer prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch DNA Wash Buffer)
- For the 50-prep kit, add 20 mL of ethanol to the Monarch DNA Wash Buffer
- For the 250-prep kit, add 100 mL of ethanol to the 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 (~13.000 rpm ).
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 | B | C | |
| 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).
Note
A 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.
Insert column into collection tube and load sample onto column. Spin at 16000 x g for 00:01:00 , then discard flow-through.
Re-insert column into collection tube. Add 200 µL DNA Wash Buffer and spin at 16000 x g for 00:01:00 .
Note
Discarding flow-through is optional.
Repeat Step 3: Re-insert column into collection tube. Add 200 µL DNA Wash Buffer and spin at 16000 x g for 00:01:00 .
Note
Discarding flow-through is optional.
Transfer column to a clean 1.5 ml microfuge tube.
Note
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 00:01:00 .
Add ≥ 6 µL DNA Elution Buffer to the center of the matrix. Wait for 00:01:00 .
Spin for 00:01:00 to elute DNA.
Note
Typical elution volumes are 6 µL –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.