Product Class: Kit

Monarch® Spin gDNA Extraction Kit

Effective August 26, 2024, product name modified to Monarch® Spin gDNA Extraction Kit. The component/product name of Monarch gDNA Purification Columns has changed to Monarch Spin Columns S2C, and these columns are now tinted orange.

Product Introduction


  • Extract and purify genomic DNA from a broad range of sample types, such as cells, blood, tissue, bacteria, yeast, saliva, swabs with just this single kit. Suitable for clinically-relevant samples (e.g., saliva, cheek).
  • Isolate high molecular weight gDNA (peak size typically ≥ 50 kb) for use directly in downstream applications, including PCR, qPCR/RT-qPCR, NGS and sequencing.
  • Obtain excellent yields of highly-pure DNA with very low residual RNA contamination (typically <1%) with included RNase A and Proteinase K.
  • Protocol compatible for cleanup of gDNA from other purifications.
  • Kit components and columns also offered separately.
  • For HMW gDNA extraction up to Mb range for long-read sequencing, see related Monarch HMW DNA Extraction Kit for Cells & Blood (NEB #T3050) or Monarch HMW DNA Extraction Kit for Tissue (NEB #T3060).

Check out our Technical Note containing comprehensive insights into measuring and analyzing nucleic acids.

Catalog # Size Concentration
T3010S 50.0 preps
T3010L 150.0 preps

Product Information

Description

The Monarch Spin gDNA Extraction Kit is a comprehensive solution for cell lysis, RNA removal, and purification of intact genomic DNA (gDNA) from a wide variety of biological samples, including cultured cells, blood, and mammalian tissues. Additionally, bacteria and yeast can be processed with extra steps to enhance lysis in these tough-to-lyse samples. Protocols are also included to enable purification from clinically-relevant samples such as saliva and cheek swabs as well as rapid cleanup of previously extracted gDNA. Purified gDNA has high quality metrics, including A260/A280 > 1.8 and A260/A230 > 2.0, high DIN scores and minimal residual RNA. The purified gDNA is suitable for downstream applications such as end-point PCR, qPCR and library prep for NGS. It typically has a peak size of > 50kb, making this kit an excellent choice upstream of long-read sequencing platforms.

Workflow for the Monarch Spin gDNA Extraction Kit




Specifications:

Input:

Cultured mammalian cells: up to 5 x 106 cells

Mammalian whole blood: 100 µl

Tissue: up to 25 mg, depending on tissue type

Bacteria: up to 2 x 109

Yeast: up to 5 x 107

Saliva: up to 500 µl

Buccal swabs

Genomic DNA requiring cleanup

Binding Capacity: 30 µg genomic DNA
Yield: Varies depending on sample type
Genomic DNA Size: Peak size > 50 kb for most sample types; may be lower for saliva and buccal swabs
RNA Content < 1% (with included RNase A treatment)
Elution Volume ≥35 µl, but 100 µl is recommended
Purity: A260/280 ≥ 1.8
A260/230 ≥ 2.0

 

Validated Sample Types:

  • Mouse Tail
  • Mouse Ear
  • Mouse Liver
  • Rat Liver
  • Mouse Kidney
  • Mouse Spleen
  • Mouse Heart
  • Mouse Lung
  • Mouse Brain
  • Rat Brain
  • Mouse Muscle
  • Rat Muscle
  • Deer Muscle
  • Human Blood
  • Mouse Blood
  • Rabbit Blood
  • Pig Blood
  • Guinea Pig Blood
  • Cow Blood
  • Horse Blood
  • Dog Blood
  • Chicken Blood
  • HeLa Cells
  • HEK293 Cells
  • NIH3T3 Cells
  • E. coli
  • Rhodobacter sp.
  • B. cereus
  • T. kodakarensis
  • S. cerevisiae
  • Saliva
  • Buccal swab


Figure 1: The Monarch Spin gDNA Extraction Kit efficiently purifies high-quality, high molecular weight gDNA from a variety of sample types.



100 ng of genomic DNA from each sample was loaded on a 0.75% agarose gel. gDNA was isolated following the standard protocols for blood, cultured cells and tissue, and the supplemental protocols for buccal swabs, saliva, Gram– and Gram+ bacteria. Starting material used: 1 x 106 HeLa cells, 100 μl human blood, 10 μl bird blood, 10 mg frozen tissue powder, 1 buccal swab, 500 μl saliva and ~1 x 109 bacterial cells. Lambda DNA-Hind III digest (NEB #N3012) was used as a marker in the last lane (M). Purified gDNA samples were analyzed using a Genomic DNA ScreenTape® on an Agilent Technologies® 4200 TapeStation®. Samples typically yield peak sizes 50–70 kb and DINs of ~9. The cell fractions processed in the buccal swab and saliva preps contain dead cells, as expected, causing a smear like pattern with typical low molecular weight apoptotic bands.


Figure 2: The Monarch Spin gDNA Extraction Kit provides excellent yields for tissues that are problematic for other commercial kits.



Duplicate 10 mg samples of RNAlater®-stabilized rat tissue were cut to small pieces and subsequently lysed and purified according to the protocols provided for each kit. Optional RNase A steps were included. Elution was carried out with 100 µl elution buffer provided in the respective kits. Yields displayed are averages of the duplicate samples, and represent the genomic DNA yield after correcting for the RNA content as determined by LC-MS. Results indicate that the Monarch Spin gDNA Extraction Kit provides excellent yields for a wide range of tissues.


Figure 3: Genomic DNA purified with the Monarch Spin gDNA Extraction Kit is high quality and suitable for sensitive applications like long range PCR and qPCR.



A. Amplification reactions were set up with primer pairs specific for 6, 8, 10, 12, 16, 20 kb amplicons from human DNA. LongAmp® Hot Start Taq 2X Master Mix (NEB #M0533) was used and 25 ng template DNA was added to each sample. PCR reactions were carried out on an Applied Biosystems 2720 Thermal Cycler. Monarch-purified genomic DNA isolated from HeLa cells and human blood were compared to commercially available reference DNA from the human cell line NA19240 F11. 10 of 20 µl was loaded on a 1.5% agarose gel, using the 1 kb DNA Ladder (NEB #N3232) as a marker. Results indicated DNA was of high-integrity and suitable for long range PCR.

B. Monarch-purified genomic DNA from human whole blood, HeLa cells and mouse tail was diluted to produce a five log range of input template concentrations. The results were generated using primers targeting gHEME (human whole blood) and gREL (HeLa, mouse tail) for qPCR assays with the Luna Universal qPCR Master Mix (NEB #M3003) and cycled on a Bio-Rad® CFX Touch qPCR thermal cycler. Results indicated that DNA is highly pure and free from inhibitors, optimal for qPCR.


Figure 4: The Monarch Spin gDNA Extraction Kit produces excellent input material for NGS library preparation with NEBNext® kits for Illumina®.



A. Duplicate libraries were made from 100 ng HeLa cell gDNA purified with Monarch (orange) or Qiagen DNeasy Mini Kit (blue) using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (NEB #E7805). Libraries were sequenced on an Illumina MiSeq. Reads were mapped using Bowtie 2.2.4 and GC coverage was calculated using Picard’s CollectGCBiasMetrics (v1.117). Expected normalized coverage of 1.0 is indicated by the horizontal grey line, the number of 100 bp regions at each %GC is indicated by the vertical grey bars, and the colored lines represent the normalized coverage for each library. Monarch GC coverage matched Qiagen DNeasy results.

B. Yields of libraries produced by enzymatic shearing are equivalent when using Monarch-purified gDNA. Library yields of the samples described above were assessed on an Agilent Technologies® 2100 BioAnalyzer using a High Sensitivity DNA Kit.


Figure 5: DNA isolation from higher volume and amounts of blood using RBC Lysis based protocol for Monarch Spin gDNA Extraction Kit

DNA extracted using RBC lysis method achieves similar high yields and concentrations as direct methods, while allowing for extraction from higher volumes and amounts of blood. Genomic DNA samples were purified in triplicate from 3 days old fresh pig blood. For the Direct Lysis samples, the DNA from 100 µl pig blood was purified using the standard protocol for fresh mammalian blood from the NEB #T3010 kit. For the RBC Lysis samples, the indicated amount of pig blood was used in the protocol for up to 500 µl blood using the NEB #T3010 kit and RBC Lysis Buffer (NEB #T3051).


Figure 6: The Monarch Spin gDNA Extraction Kit generates high quality DNA for Nanopore sequencing.

Moarch Human gDNA HeLa

HeLa cell genomic DNA was extracted using either the Monarch Spin gDNA Extraction Kit or the Qiagen DNeasy Blood & Tissue Kit. One microgram of purified DNA was used to prepare Oxford Nanopore Technology (ONT) sequencing libraries following the ONT 1D Ligation Sequencing Kit (SQK-LSK109) protocol without DNA fragmentation. Libraries were loaded on a GridION (Flow cell R9.4.1) and the data was collected for 48 hrs. Libraries produced using the Monarch gDNA exceeded the Qiagen libraries on common sequencing metrics including: A. total sequencing data collected, B. read quality, and C. read length. Data was generated using NanoComp (Bioinformatics, Volume 34, Issue 15, 1 August 2018, Pages 2666–2669).


This product is related to the following categories:
Genomic DNA Extraction & Purification Products,
Nucleic Acid Purification Products,
This product can be used in the following applications:
Nucleic Acid Purification

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Product Notes

  1. Monarch RNase A and Proteinase K should be stored at -20°C after opening the kit.

Protocols, Manuals & Usage

Protocols

  1. Quick Protocol for Extraction and Purification of Genomic DNA
  2. Protocol for Extraction and Purification of Genomic DNA from Cells (NEB #T3010)
  3. Protocol for Extraction and Purification of Genomic DNA from Tissues (NEB #T3010)
  4. Protocol for Extraction and Purification of Genomic DNA from Blood (NEB #T3010)
  5. Genomic DNA Extraction from Gram-negative Bacteria (NEB #T3010)
  6. Genomic DNA Extraction from Gram-positive Bacteria and Archaea (NEB #T3010)
  7. Genomic DNA Extraction from Yeast (NEB #T3010)
  8. Genomic DNA Extraction from Buccal Swabs (NEB #T3010)
  9. Genomic DNA Extraction from Saliva (NEB #T3010)
  10. Protocol for Genomic DNA Cleanup (NEB #T3010)
  11. Genomic DNA Extraction from Insects
  12. Protocol for Isolation of Genomic DNA (gDNA) from up to 500 µl mammalian blood using the Monarch® Spin gDNA Extraction Kit (NEB #T3010) and RBC Lysis Buffer (NEB #T3051)
  13. Protocol for Genomic DNA Extraction from Cultured Cells Stabilized with Monarch® DNA/RNA Protection Reagent (NEB #2011) using the Monarch Spin gDNA Extraction Kit (NEB #T3010)
  14. Protocol for Genomic DNA Extraction from Blood Stabilized with Monarch® DNA/RNA Protection Reagent (NEB #T2011) using the Monarch Spin gDNA Extraction Kit (NEB #T3010)
  15. Protocol for Genomic DNA Extraction from Tissue Samples Stabilized with Monarch® DNA/RNA Protection Reagent (NEB #T2011) using the Monarch Spin gDNA Extraction Kit (NEB #T3010)
  16. Protocol for Genomic DNA Extraction from Gram-negative Bacteria using NEBExpress® T4 Lysozyme (NEB #P8115) and the Monarch Spin gDNA Extraction Kit (NEB #T3010)
  17. Protocol for Genomic DNA Extraction from Gram-positive Bacteria using NEBExpress® T4 Lysozyme (NEB #P8115) and the Monarch Spin gDNA Extraction Kit (NEB #T3010)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Usage & Guidelines

Application Notes

FAQs & Troubleshooting

FAQs

  1. What is the composition of each buffer provided with the Monarch® Spin gDNA Extraction Kit?
  2. What is the maximum binding capacity of the Monarch® Spin gDNA Extraction Kit columns?
  3. What is the minimum elution volume that can be used with the Monarch® Spin gDNA Extraction Kit columns?
  4. How important is it to pre-heat the elution buffer to 60°C? Would 56°C be OK?
  5. Can I get better recovery with the Monarch® Spin gDNA Extraction Kit if I do a second elution with my eluent from the first elution?
  6. Can I use a different elution buffer than the one supplied with the Monarch® Spin gDNA Extraction Kit (NEB #T3010)?
  7. What size gDNA can be purified with the Monarch® Spin gDNA Extraction Kit?
  8. What is the minimal input amount that can be used with the Monarch® Spin gDNA Extraction Kit?
  9. Can I use the Monarch® Spin gDNA Extraction Kit to clean up gDNA that was extracted with phenol/chloroform?
  10. Can I use the Monarch® Spin gDNA Extraction Kit for gDNA cleanup?
  11. How can I assess gDNA integrity and purity?
  12. Can I use a different RNase A with the Monarch® Spin gDNA Extraction Kit? 
  13. Can I use a different Proteinase K with the Monarch® Genomic DNA Purification Kit?
  14. Can I omit the low-speed binding centrifugation step to save time?
  15. Can I omit the RNase A digestion step from the protocol? 
  16. How should I store purified gDNA samples?
  17. Is Monarch-purified gDNA suitable for long-range PCR?
  18. Can I purchase Monarch® buffers and columns separately?
  19. When working with other commercial silica column-based kits, I occasionally see a white precipitate in the eluate. What is this?
  20. Can I use Monarch® RNase A for applications/workflows other than gDNA purification?
  21. Is Monarch-purified gDNA compatible with Next Generation Sequencing (NGS) platforms like PacBio and nanopore?
  22. Why did the silica membrane on my Monarch Spin Columns S2C (NEB #T3017) dislodge during my genomic DNA prep?
  23. Can the Monarch® Spin gDNA Extraction Kit (NEB #T3010) be used to isolate gDNA from drosophila and other insects?
  24. Can the Monarch® Spin gDNA Extraction Kit (NEB #T3010) be used to isolate gDNA from plants?
  25. Can the Monarch® Spin gDNA Extraction Kit (NEB #T3010) be used to isolate gDNA from plasma, serum and urine?
  26. Can the Monarch® Spin gDNA Extraction Kit (NEB #T3010) be used for isolating cfDNA?
  27. Can the Monarch® Spin gDNA Extraction Kit (NEB #T3010) be used with a vacuum manifold?
  28. The enzymes in the kit were not stored at -20°C right away. Will they still work?
  29. Does it matter which anticoagulant is used in blood samples?
  30. There is a precipitate in the Proteinase K enzyme tube, is this normal?
  31. I notice that I'm applying force to fit the column into a collection tube. Should I be doing this? What collection tubes are compatible with columns?
  32. I noticed that in the Monarch® Spin gDNA Extraction Kit (NEB #T3010), the Monarch Spin Columns S2C (NEB #T3017) for gDNA purification are now tinted orange. Does this impact performance?

Troubleshooting

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specification Change Notifications

Effective August 26, 2024, product name modified to Monarch®Spin gDNA Extraction Kit. The component/product name of Monarch gDNA Purification Columns has changed to Monarch Spin Columns S2C, and these columns are now tinted orange.

Effective March 14, 2024: Proteinase K, Molecular Biology Grade component number has changed with the concentration changing from 800U/ml to 20 mg/ml.

Effective May 9, 2022: Shelf life is 24 months.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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