Description
The Monarch Spin High-Capacity DNA Cleanup Kit (100 μg) offers class-leading binding capacity and proven Monarch buffer chemistry, while maintaining the convenient spin column format for silica membrane-based DNA cleanup. This is a high-capacity version of our Monarch Spin PCR & DNA Cleanup Kit (5 μg) (NEB #T1130). The Monarch Spin High-Capacity DNA Cleanup Kit (100 μg) efficiently purifies and concentrates up to 100 μg of high-quality, double-stranded and single-stranded DNA from enzymatic reactions such as RCA, RE digestion, labeling, or exonuclease treatment. The high capacity and rapid process time make the kit an excellent choice for cleanup of templates generated by cell-free DNA synthesis workflows. Modified protocols enabling cleanup of 100 µg amounts of oligonucleotides, genomic DNA, or RCA products make this kit a workhorse for multiplexed, high-throughput workflows requiring large amounts of starting materials. The kit features precision-engineered spin columns uniquely designed to enable low elution volumes (50 μl), resulting in highly concentrated DNA suitable for downstream applications such as in vitro transcription (IVT), genome editing, or viral vector transfection.
The kit includes spin columns, collection tubes, buffers, and other reagents, all compactly packaged. The spin columns are compatible with microcentrifuges, centrifuges, and vacuum manifolds, without additional plastic accessories. Manufactured using state-of-the-art automation, Monarch spin columns ensure high quality and consistency. The optimized buffer system helps ensure efficient DNA binding and purification, without the need to adjust pH.
Monarch kits are more eco-friendly than other options from leading suppliers, reducing plastic usage in the columns and bottles while maintaining structural integrity for centrifugation or vacuum manifolds.
| Properties |
| Purification format |
Spin column |
| DNA Sample |
DNA from PCR and other enzymatic reactions (e.g., restriction digests, RCA, labeling, exonuclease treatment, ligations) |
ssDNA or dsDNA oligonucleotides from enzymatic reactions can also be purified using the Oligonucleotide Cleanup Protocol
|
Genomic DNA and large size DNA (>25 kb) or RCA reaction cleanup using the supplemental protocols
|
| Typical Recovery |
70-90% |
| DNA Purity |
A260/280 > 1.8 and A260/230 > 1.8 |
| Nucleic Acid Binding Capacity |
Up to 100 μg of DNA |
| Elution Volume |
50-200 μl |
| DNA Size Range |
Standard protocol: 40 bp – 25 kb |
Oligonucleotide Cleanup protocol: ssDNA >12 nt and dsDNA >10 nt
|
| Protocol Time |
9 minutes of spin and incubation time |
| Compatible Downstream Applications |
Ligation, in vitro transcription (IVT), genome editing, viral transfection, restriction digestion, labeling and other enzymatic manipulations, library construction and DNA sequencing
|
Figure 1: Monarch Spin High-Capacity DNA Cleanup Kit (100 µg) offers a wide range of DNA input amounts
DNA purifications were performed using the Monarch Spin High-Capacity DNA Cleanup Kit (100 µg) with varying amounts of DNA. To assess average DNA recovery, DNA input (1 kb Ladder, NEB #N3232) ranging from 10 to 100 µg was used and eluted in 100 µl of Monarch Buffer EY, in triplicate. DNA concentrations for both input and eluted samples were measured using a Trinean DropSense 16. Percent recovery calculations were based on the measured DNA concentrations and the elution volume.
Figure 2: Monarch Spin High-Capacity DNA Cleanup Kit (100 µg) effectively recovers various DNA sample types, maximizing utility of the kit
DNA samples were purified in triplicate using the Monarch Spin High-Capacity DNA Cleanup Kit (100 µg), following either the standard protocol (for linear, circular, and ssDNA) or the supplemental protocol (for large DNA). Each sample, except for the RCA product (50 µl, 1 mg/ml), contained 10 µg of DNA and was eluted in 100 µl of Monarch Buffer EY. DNA concentrations for both input and eluted samples were measured using a Trinean DropSense 16, except for the RCA product, which was measured using the Qubit dsDNA Broad Range (BR) Assay Kit. Percent recovery was calculated based on the measured DNA concentrations and elution volumes, with results displayed in a bar graph. For linear DNA, the following ladders were utilized: LMW DNA ladder (25-766 bp, NEB #N3233), 1 kb DNA Ladder (0.5–10 kb, NEB #N3232), and HindIII digest (2–23 kb, NEB #N3012). For circular DNA, pUC19 (NEB #N3041), PhiX174 RF I (NEB #N3021) and PhiX174 RF II (NEB #N3022) were employed. For ssDNA, phiX174 Virion (NEB #N3023) and synthetic ssDNA (150 nt) were used.
Figure 3: Monarch Spin High-Capacity DNA Cleanup Kit (100 µg) effectively recovers oligonucleotide DNA using the oligonucleotide cleanup protocol
DNA samples were purified in triplicate using the Monarch Spin High-Capacity DNA Cleanup Kit (100 µg) with the provided oligonucleotide cleanup protocol. To assess average oligonucleotide recovery, 10 µg of synthesized ssDNA (8–25 nt) and dsDNA (8–25 bp) were used and eluted in 100 µl of Monarch Buffer EY. DNA concentrations for both input and eluted samples were measured using a Trinean DropSense 16. Percent recovery was calculated based on the measured DNA concentrations and elution volumes, with results displayed in a bar graph. Using this protocol, ssDNA (≥ 12 nt) and dsDNA (10 bp) can be effectively recovered.
Figure 4: The Monarch Spin High-Capacity DNA Cleanup Kit (100 µg) efficiently removes Proteinase K, demonstrating protein removal from enzymatic reactions
DNA samples containing 0.8 units or 8 units of Proteinase K (NEB #P8200SVIAL) were purified using the Monarch Spin High-Capacity DNA Cleanup Kit (100 µg). The eluted DNA was then tested for residual Proteinase K by incubating 10 µg MBP5 in 1X NEBuffer 4 at 37°C for 5 minutes. A proteinase K control titration (0.002-3.12 x10-5 units, 2-fold dilution, and 0 units) was used as a control, as shown in the left gel. The right gel represents six replicate purified samples with 0.8 units or 8 units of Proteinase K in the input sample, showing that MBP5 remains undigested at 42.5 kDa.
Figure 5: DNA purified with the Monarch Spin High-Capacity DNA Cleanup Kit (100 µg) is of high quality and ideal for downstream applications
To demonstrate downstream application compatibility, an in vitro transcription (IVT) reaction was performed using a DNA template purified with the Monarch Spin High-Capacity DNA Cleanup Kit (100 µg). The pCMV-CLuc 2 plasmid or RCA product, using pCMV-CLuc 2 as a template, was linearized with NotI-HF (NEB #R3189). The linearized DNA was then purified using the Monarch Spin High-Capacity DNA Cleanup Kit (100 µg), resulting in highly pure DNA, as indicated by the A260/230 and A260/280 ratios. This purified DNA served as the template for the IVT reaction, using the HiScribe® T7 High Yield RNA Synthesis Kit (NEB #E2040). The reaction was scaled to 100 µl, with a FLuc IVT reaction performed as a control using the FLuc Control Template provided in the HiScribe T7 High Yield RNA Synthesis Kit (NEB #E2040).
Figure 6: Overview of the Monarch Spin High-Capacity DNA Cleanup Kit (100 µg) workflow
Figure 7: Features of the Monarch Spin Column S3A for High-Capacity DNA Cleanup
- This product is related to the following categories:
-
PCR & DNA Cleanup,
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Nucleic Acid Purification,
- This product can be used in the following applications:
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Nucleic Acid Purification