Product Class: Kit

Monarch® HMW DNA Extraction Kit for Cells & Blood


Catalog #T3050

Product Introduction

  • Designed for extraction for long-read sequencing applications from cells and blood. See related Monarch Genomic DNA Purification Kit (NEB #T3010) for gDNA extraction for general NGS and sequencing (Illumina® and other).
  • Isolate high molecular weight DNA (100 kb to megabase range) for Oxford Nanopore®, PacBio® sequencing
  • Uses unique, innovative technology for fast, user-friendly workflows, only 30-60 minutes for extraction compared to hours or days.
  • Includes RNase A and Proteinase K for effective RNA removal and digestion of cellular proteins
  • Obtain best-in-class yields of highly-pure and intact DNA
  • For HMW gDNA extraction from tissues, bacteria, and more for long-read sequencing, see related Monarch HMW DNA Extraction Kit for Tissue (NEB #T3060)

Featured Resources:

  • Check out our publication in Biotechniques outlining a simple approach to accurate quantitation of ultra high molecular weight (UHMW) DNA.
  • Review our technical note examining the use and comparison of a novel method to isolate genomic DNA from bacteriophages.

Product Information

Description

The Monarch HMW DNA Extraction Kit for Cells & Blood provides a rapid and reliable process for extracting high molecular weight (HMW), intact genomic DNA from cultured cells and whole blood. Utilizing an optimized process that combines gentle cell lysis with a tunable fragment length generation followed by precipitation of the extracted DNA onto the surface of large glass beads, the prep proceeds rapidly and utilizes standard laboratory equipment. DNA size ranges from 50-250 kb for the standard protocol and into the Mb range when the lowest agitation speeds are used. Purified DNA is recovered in high yield with excellent purity, including nearly complete removal of RNA. For cells, the process time is only 30 min, while blood samples require erythrocyte lysis and are processed in 60 min. Purity ratios of 1.80-1.90 and 2.2-2.5 (A260/A280 and A260/A230, respectively), are easily and reproducibly achievable, and purified HMW DNA is suitable for a variety of downstream applications including long-read sequencing (Oxford Nanopore Technologies® and Pacific Biosciences®), optical mapping (Bionano Genomics®), and linked-read genome assembly.

Validated Sample Types

Cells

  • K293
  • HeLa
  • NIH3T3
  • Jurkat
  • K562 (suspension cells)
  • HCT116
  • A549
  • U5Os
  • HepG2
  • NCI-460
  • SK-N-SH
  • Aa23

Blood

  • Human
  • Mouse
  • Rat (fresh only)
  • Rabbit

 

 

  • Pig
  • Horse
  • Cow
  • Rhesus money
  • Goat (fresh only)
  • Sheep (fresh only)
  • Chicken
  • Turkey

Visit our input guidelines or more information

 

 

Figure 1: Workflow for isolation of HMW DNA from cells using the Monarch HMW DNA Extraction Kit






Figure 2: Workflow for isolation of HMW DNA from blood using the Monarch HMW DNA Extraction Kit






Figure 3: Reproducible Extraction of HMW DNA from Cells and Blood with the Monarch HMW DNA Extraction Kit




DNA extracted with Monarch HMW DNA Extraction Kit for Cells & Blood. 1 x 106 fresh HEK293 cells and 500 µl fresh human blood were used as inputs and for preps performed according to the kit instructions using the agitation speed indicated above the gel lanes. 500 ng of DNA from the replicates was resolved by PFGE (1% agarose gel, 6 V/cm, 13°C for 20 hours, switch times ramped from 0.5–94 seconds on a BioRad® CHEF-DR® III System). Yield and purity ratios of the individual preps are shown in the accompanying tables. Lambda PFG Ladder (NEB #N0341) was used as molecular weight standard.





Figure 4: Use of varying agitation speeds during lysis produces tunable fragment length of extracted HMW genomic DNA from cells and blood




Preps were performed on duplicate aliquots of 1 x 106 HEK 293 cells and 500 ìl fresh human blood. Samples were agitated at the indicated speed during the lysis step to control the fragmentation of the DNA. Equal amounts of DNA from the replicates (cells: 500 ng; blood: 650 ng) were resolved by PFGE (1% agarose gel, 6 V/cm, 13°C for 20 hours, switch times ramped from 0.5–94 seconds on a BioRad CHEF-DR III System). Yield and purity ratios of the individual preps are shown in the accompanying tables. Lambda PFG Ladder and Lambda DNA-Hind III Digest (NEB #N0341 and #N3012) were used as molecular weight standards. Yield, purity ratios and DINs of the individual preps are shown in the accompanying tables.





Figure 5: Linear correlation between DNA yield and input for cell and blood samples




Summarized yield data for HMW DNA preps are shown carried out at 2,000 rpm during lysis, using HEK293 cultured cells and fresh human blood samples from different donors as input material in the corresponding protocols. The starting materials were diluted to 5 different concentrations to cover the entire recommended input range. Cell samples ≤ 5x105 cells and blood samples <500 µl were purified using the recommended volumes for low input samples. Obtained yields show a high degree of linearity over the displayed input range.





This product is related to the following categories:
Genomic DNA Extraction & Purification Products,
Nucleic Acid Purification Products,

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Download Quick Protocol Card for Cells
  2. Download Quick Protocol Card for Blood
  3. Protocol for High Molecular Weight DNA (HMW DNA) Extraction from Cells (NEB #T3050)
  4. Protocol for High Molecular Weight DNA (HMW DNA) Extraction from Blood (NEB #T3050)
  5. Protocol for UHMW DNA Cleanup in the Oxford Nanopore Technologies® UL Library Prep Workflow
  6. Protocol Guidance for Extraction of Ultra-High Molecular Weight (UHMW) Genomic DNA for Ultra-Long (UL) Read NGS Sequencing applications in Oxford Nanopore Technologies® workflows

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. How does excess residual protein in blood and tissue preps affect the purification process?
  2. Which measurement system is best used for assessing the concentration of HMW DNA?
  3. I dropped a few of my glass beads. Can I clean them, and do you supply extra?
  4. Is there anything special about the Monarch DNA Capture Beads, or can I use any glass beads?
  5. Are the Monarch HMW DNA Extraction kits compatible with samples stored in Monarch DNA/RNA Protection Reagent, RNAlater, DNA/RNA Shield etc.? 
  6. How do I know that my HMW DNA has attached to glass beads in the Monarch HMW DNA Extraction Kits?
  7. Is there any benefit to adding in a size selection (e.g., Circulomics® Short Read Eliminator) following extraction?
  8. Can I still use the Monarch HMW DNA Extraction Kits if I do not have a thermal mixer?
  9. Can I use the Monarch HMW DNA Extraction Kits for processing plant samples?
  10. Can I use the Monarch HMW DNA Extraction Kits for processing insect samples?
  11. Can the Monarch HMW DNA Extraction Kit be used for processing yeast and/or fungal samples?
  12. Can the Monarch HMW DNA Extraction Kits be used to process human Buffy coat?
  13. Can the Monarch HMW DNA Extraction Kits be used to process mouthwash samples?
  14. Can the Monarch HMW DNA Extraction Kits be used to process environmental or fecal samples for metagenomic studies?
  15. Can the Monarch HMW DNA Extraction Kits be used to isolate BAC DNA?
  16. Do you have any PacBio Sequencing results from DNA extracted from tissue samples with the Monarch HMW DNA Extraction Kits?
  17. Does the Monarch HMW DNA Extraction workflow work for single cells?
  18. Can you use the DNA isolated with the Monarch HMW DNA Extraction Kits in Southern blots?
  19. Can the Monarch HMW DNA Extraction Kits be used to process nematodes?
  20. My blood sample is frozen in aliquots larger than is recommended in your protocol (e.g., in Vacutainers). Can I still use the frozen blood protocol for the Monarch HMW DNA Extraction Kit for Cells & Blood (NEB #T3050)?
  21. I am having trouble dissolving my HMW DNA after isolation. Do you have any tips on increasing the solubility?
  22. The enzymes in the kit were not stored at -20°C right away. Will they still work?
  23. There is a precipitate in the Proteinase K enzyme tube, is this normal?
  24. Which Monarch® columns and bead retainers can be used with which Monarch collection tube?
  25. I noticed the collection tube dimensions has changed slightly in the Monarch® HMW DNA Extraction Kit for Cells & Blood (NEB #T3050) and the Monarch HMW DNA Extraction Kit for Tissue (NEB #T3060). Will this impact performance?
  26. 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?

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 March 18,2024, collection tube dimensions modified and component numbers changed.

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

 

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]

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.

Licenses

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 gbd@neb.com While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
 

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