Monarch® HMW DNA Extraction Kit for Tissue |NEB酶试剂 New England Biolabs

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产品信息

The Monarch HMW DNA Extraction Kit for Tissue provides a rapid and reliable process for extracting high molecular weight (HMW), intact genomic DNA from various tissues and bacteria, as well as other sample types including yeast, insect, and amphibian.  The optimized extraction protocol for tissue utilizes pestle homogenization and proteinase K digestion with agitation for sample lysis, followed by a protein removal step and precipitation of the extracted DNA onto the surface of large glass beads.  A slightly modified extraction protocol for bacteria utilizes lysozyme for the efficient lysis of the bacterial cell wall, prior to proteinase K digestion.  DNA size ranges from 50- ≥500 kb for the standard protocols and into the Mb range when the lowest agitation speeds are used for processing soft organ tissues and bacteria. Purified DNA is recovered in high yield with excellent purity, including nearly complete removal of RNA. For tissue and bacteria, the processing time is 90 min. Purity ratios are typically 1.8-1.9 (260/280) for tissue and bacteria, 2.1-2.5 (260/230) for tissue, and 2.1-2.2 (260/230) for bacteria.  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

  • Mouse brain
  • Mouse liver
  • Mouse muscle
  • Mouse kidney
  • Mouse tail
  • Mouse ear punch
  • Rat kidney
  • E. coli
  • B. cereus
  • M. luteus
  • X. laevis
  • S. cerevisiae
  • C. elegans
  • A. aegypti

Visit our input guidelines for more information

 

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

Monarch® HMW DNA Extraction Kit for Tissue | 

Figure 2: The Monarch HMW DNA Extraction Kit for Tissue efficiently purifies high-quality, HMW DNA from a variety of sample types

Monarch® HMW DNA Extraction Kit for Tissue |

HMW genomic DNA extracted with the Monarch HMW DNA Extraction Kit for Tissue. 10 mg frozen rat kidney, 10 mg fresh mouse liver, 20 mg frozen mouse brain, 20 mg fresh mouse muscle, 2 x 109 frozen E. coli cells, 2 x 108 frozen B. cereus cells, 4 mg fresh X. laevis, 3.8 x 108 fresh S. cerevisiae cells and 15 mg frozen A. aegypti were used as inputs for preps. Preps were performed according to the kit instructions with sample agitation at 2000 rpm. A modified workflow was used to process S. cerevisiae preps. 500 ng of DNA from each sample prep 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 a molecular weight standard.

Figure 3: Use of varying agitation speeds during lysis produces tunable fragment length for HMW genomic DNA extracted from soft organ tissues and bacteria

Monarch® HMW DNA Extraction Kit for Tissue |

HMW genomic DNA from mouse kidney (10 mg, Figure 3A) and E. coli NEB10 beta (1 x 109 cells, Figure 3B) was purified using the Monarch HMW DNA Extraction Kit for Tissue. Samples were agitated at the indicated speed during the lysis step to control the fragmentation of the DNA. 300 ng (kidney) and 500 ng (E. coli) of DNA for each sample was resolved by PFGE (settings: switch time 0.5 to 94 sec; run time 20 hours at 6 V/cm, angle 120°, temperature 13°C on the BioRad CHEF-DR III System). Lambda PFG Ladder (NEB #N0341) was used as molecular weight standard. Yield, purity ratios and DIN values (kidney) of the individual preps are shown in the accompanying tables

产品类别:
Genomic DNA Extraction & Purification Products,
Nucleic Acid Purification Products

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • T3060S     25    
        Monarch® HMW gDNA Tissue Lysis Buffer T3061-21 25 1 x 3.5 ml Not Applicable
        Monarch® Protein Separation Solution T3062-21 25 1 x 2 ml Not Applicable
        Proteinase K, Molecular Biology Grade P8200GVIAL -20 1 x 0.11 ml 20 mg/ml
        Monarch® RNase A T3018-21 -20 2 x 0.035 ml 20 mg/ml
        Monarch® gDNA Wash Buffer T3015-21 25 1 x 4.5 ml Not Applicable
        Monarch® gDNA Elution Buffer II T3056-21 25 1 x 1.5 ml Not Applicable
        Monarch® DNA Capture Beads T3005-21 25 1 x 10 beads Not Applicable
        Monarch® Pestle Tubes T3001-21 25 1 x 5 tubes Not Applicable
        Monarch® Pestles T3002-21 25 1 x 5 pestles Not Applicable
        Monarch® 2 ml Tubes T3003-21 25 1 x 10 tubes Not Applicable
        Monarch® Bead Retainers T3004-21 25 1 x 5 items Not Applicable
        Monarch® Spin Collection Tubes T2118-23 25 1 x 5 tubes Not Applicable
    • T3060L     25    
        Monarch® HMW gDNA Tissue Lysis Buffer T3061-1 25 1 x 31 ml Not Applicable
        Monarch® Protein Separation Solution T3062-1 25 1 x 18 ml Not Applicable
        Proteinase K, Molecular Biology Grade P8200SVIAL -20 1 x 1 ml 20 mg/ml
        Monarch® RNase A T3018-2 -20 1 x 0.5 ml 20 mg/ml
        Monarch® gDNA Wash Buffer T3015-1 25 1 x 18 ml Not Applicable
        Monarch® gDNA Elution Buffer II T3056-1 25 1 x 12 ml Not Applicable
        Monarch® DNA Capture Beads T3005-1 25 1 x 100 beads Not Applicable
        Monarch® Pestle Tubes T3001-1 25 1 x 50 tubes Not Applicable
        Monarch® Pestles T3002-1 25 1 x 50 pestles Not Applicable
        Monarch® 2 ml Tubes T3003-1 25 2 x 50 tubes Not Applicable
        Monarch® Bead Retainers T3004-1 25 1 x 50 items Not Applicable
        Monarch® Spin Collection Tubes T2118-1 25 1 x 50 tubes Not Applicable

  • 相关产品

    相关产品

    • Monarch 高分子量 DNA 提取试剂盒（细胞和血液）
    • T3000 Monarch Pestle Set
    • Monarch DNA 捕获珠
    • Monarch 2 ml 离心管
    • Monarch 捕获珠分离管
    • T3061 Monarch HMW gDNA Tissue Lysis Buffer
    • T3062 Monarch Protein Separation Solution
    • T3056 Monarch gDNA Elution Buffer II

操作说明、说明书 & 用法

  • 操作说明

    1. Download Quick Protocol Card for Tissue
    2. Protocol for High Molecular Weight DNA (HMW DNA) Extraction from Tissue (NEB #T3060)
    3. Protocol for High Molecular Weight DNA (HMW DNA) Extraction from Bacteria (NEB #T3060)
    4. Protocol for High Molecular Weight DNA (HMW DNA) Extraction from Yeast (NEB #T3060)
    5. Protocol for High Molecular Weight DNA (HMW DNA) Extraction from Phage
    6. Protocol for High Molecular Weight DNA (HMW DNA) Extraction from Phage
    7. Protocol for UHMW DNA Cleanup in the Oxford Nanopore Technologies® UL Library Prep Workflow

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualT3060

  • 使用指南

    • Choosing an Agitation Speed During Lysis with the Monarch HMW DNA Extraction Kits
    • Choosing Input Amounts for the Monarch HMW DNA Extraction Kits
    • Considerations and Performance Data for Nanopore Sequencing of High Molecular Weight DNA (HMW DNA)
    • Considerations for Loading Pulsed-Field Gels (PFG) with High Molecular Weight DNA
    • Homogenization of High Molecular Weight DNA (HMW DNA) Samples After Elution
    • Important Considerations for Starting Materials When Using the Monarch HMW DNA Extraction Kit for Tissues (NEB #T3060) 
    • Measuring, Analyzing & Storing High Molecular Weight DNA (HMW DNA) Samples

  • 应用实例

    • A faster workflow for the assessment of genomic loci in mice using a novel HMW DNA extraction technology upstream of Cas9 targeted sequencing
    • Fast & efficient isolation of phage genomic DNA using the Monarch HMW DNA Extraction Kits

FAQs & 问题解决指南

  • 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 marine samples, including marine invertebrates?
    13. Can the Monarch HMW DNA Extraction Kits be used to process human Buffy coat?
    14. Can the Monarch HMW DNA Extraction Kits be used to process mouthwash samples?
    15. Can the Monarch HMW DNA Extraction Kits be used to process environmental or fecal samples for metagenomic studies?
    16. Can the Monarch HMW DNA Extraction Kits be used to isolate BAC DNA?
    17. How do you suggest homogenizing tougher tissues, like ear punches or muscle? Can a sonicator be used for tissue homogenization?
    18. Does the kit work with frozen tissue, tissue stored in preservatives (ethanol, DMSO-salt/DESS, or RNAlater®), FFPE samples, or OCT-preserved tissues?
    19. Do you see different fragmentation results when using the pestle versus the rotor-stator homogenizer for tissue homogenization?
    20. Do you have any PacBio Sequencing results from DNA extracted from tissue samples with the Monarch HMW DNA Extraction Kits?
    21. Does the Monarch HMW DNA Extraction workflow work for single cells?
    22. Can you use the DNA isolated with the Monarch HMW DNA Extraction Kits in Southern blots?
    23. Can the Monarch HMW DNA Extraction Kits be used to process nematodes?
    24. I am having trouble dissolving my HMW DNA after isolation. Do you have any tips on increasing the solubility?
    25. The enzymes in the kit were not stored at -20°C right away. Will they still work?
    26. There is a precipitate in the Proteinase K enzyme tube, is this normal?
    27. Which Monarch® columns and bead retainers can be used with which Monarch collection tube?
    28. 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?

  • 问题解决指南

    • Troubleshooting Guide for Monarch HMW DNA Extraction Kits (NEB #T3050 and #T3060)