Monarch® 高分子量 DNA 提取试剂盒（细胞和血液） |NEB酶试剂 New England Biolabs

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产品信息

Monarch 高分子量 DNA 提取试剂盒（细胞和血液）为从培养细胞和全血中提取完整的高分子量（HMW）gDNA 提供了快速可靠的操作流程。仅需使用标准实验室设备制备，即可利用优化的流程，将温和的细胞裂解过程与片段长度调节相结合，然后将提取的 DNA 沉淀到大玻璃珠表面，过程快速而高效。使用标准操作流程时，DNA 大小在 50-250 kb 之间；使用最低振荡速度时，可将 DNA 长度提高至 Mb 级。纯化后的 DNA 产量高和纯度极高，且几乎完全去除 RNA。细胞样本的提取时间仅为 30 分钟；血液样本需要裂解红细胞，可在 60 分钟内完成提取。A260/A280 在 1.80-1.90 之间，A260/A230 在 2.2-2.5 之间，并具有高度的可重复性。纯化后的高分子量 DNA 适用于多种下游应用，包括长读长测序（Oxford Nanopore Technologies® 和 Pacific Biosciences®）、光学图谱技术（Bionano Genomics®）和链读测序基因组组装。

经过验证的样本类型

细胞

  • K293
  • HeLa
  • NIH3T3
  • Jurkat
  • K562（悬浮细胞）
  • HCT116
  • A549
  • U5Os
  • HepG2
  • NCI-460
  • SK-N-SH
  • Aa23

血液

  • 人类
  • 小鼠
  • 大鼠（仅验证新鲜样本）
  • 兔

 

 

  • 猪
  • 马
  • 牛
  • 恒河猴
  • 山羊（仅验证新鲜样本）
  • 绵羊（仅验证新鲜样本）
  • 鸡
  • 火鸡

访问我们的起始量指南了解更多信息

 

 

图 1：使用 Monarch 高分子量 DNA 提取试剂盒从细胞中分离高分子量 DNA 的实验流程

Monarch® 高分子量 DNA 提取试剂盒（细胞和血液） |

图 2：使用 Monarch 高分子量 DNA 提取试剂盒从血液中分离高分子量 DNA 的实验流程

Monarch® 高分子量 DNA 提取试剂盒（细胞和血液） |

图 3：使用 Monarch 高分子量 DNA 提取试剂盒从细胞和血液中重复提取高分子量 DNA

Monarch® 高分子量 DNA 提取试剂盒（细胞和血液） |

使用 Monarch 高分子量 DNA 提取试剂盒（细胞和血液）提取 DNA。使用 1 x 106 HEK293 细胞和 500 µl 新鲜人血作为起始样本，根据试剂盒说明书提取 DNA，并按照使用凝胶泳道上方所示的速度振荡样本。从平行实验中取 500 ng DNA 通过 PFGE 分离（在 BioRad® CHEF-DR® III System 上，使用 1% 琼脂糖凝胶，6 V/cm，13℃ 电泳 20 小时，转换时间为 0.5–94 秒）。各样本的产量和纯度见附表标注。使用 Lambda PFG Ladder（NEB #N0341）作为分子量标准。

图 4：在裂解过程中使用不同的振荡速度，可调节从细胞和血液中提取的高分子量基因组 DNA 的片段长度

Monarch® 高分子量 DNA 提取试剂盒（细胞和血液） |

1 x 10^6 HEK293 细胞和 500 ìl 新鲜人血一式两份进行提取。在裂解步骤中以指定速度振荡样本，以控制 DNA 的片段化。从平行实验中取等量的 DNA 产物（细胞：500 ng；血液：650 ng）通过 PFGE 分离（在 BioRad CHEF-DR III System 上，使用 1% 琼脂糖凝胶，6 V/cm，13℃ 电泳 20 小时，转换时间为 0.5–94 秒）。各样本的产量和纯度见附表标注。使用 Lambda PFG Ladder 和 Lambda DNA-HindIII 消化（NEB #N0341 和 #N3012）作为分子量标准。各样本的产量、纯度和 DIN 值见附表标注。

图 5：细胞和血液样本的 DNA 产量和起始量呈线性相关

Monarch® 高分子量 DNA 提取试剂盒（细胞和血液） |

根据说明书操作说明，将 HEK293 培养细胞和来自不同供体的新鲜人血作为起始样本提取高分子量 DNA 并记录产量数据，裂解步骤中振荡速度为 2,000 rpm。将起始样本稀释成 5 种不同浓度，以覆盖建议的起始样本量范围。按照低起始量样本的操作说明，使用其中推荐的加液体积，从 ≤ 5 x 10^5 的细胞样本或 < 500 µl 血液样本中纯化 DNA。在测量范围内，产量与起始样本量呈高度线性相关。

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
    • T3050S     25    
        Monarch® RBC Lysis Buffer T3051-21 25 1 x 15 ml Not Applicable
        Monarch® gDNA Nuclei Prep Buffer T3052-21 25 1 x 1.5 ml Not Applicable
        Monarch® gDNA Nuclei Lysis Buffer T3053-21 25 1 x 1.5 ml Not Applicable
        Monarch® Precipitation Enhancer T3055-21 25 1 x 1 ml Not Applicable
        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
        Proteinase K, Molecular Biology Grade P8200GVIAL -20 1 x 0.11 ml 20 mg/ml
        Monarch® RNase A T3018-21 -20 1 x 0.035 ml 20 mg/ml
        Monarch® DNA Capture Beads T3005-21 25 1 x 10 beads 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
    • T3050L     25    
        Monarch® gDNA Nuclei Prep Buffer T3052-1 25 1 x 10 ml Not Applicable
        Monarch® gDNA Nuclei Lysis Buffer T3053-1 25 1 x 10 ml Not Applicable
        Monarch® Precipitation Enhancer T3055-1 25 1 x 5 ml Not Applicable
        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
        Proteinase K, Molecular Biology Grade P8200AAVIAL -20 1 x 0.55 ml 20 mg/ml
        Monarch® RBC Lysis Buffer T3051-1 25 1 x 160 ml Not Applicable
        Monarch® RNase A T3018-1 -20 2 x 0.17 ml 20 mg/ml
        Monarch® DNA Capture Beads T3005-1 25 1 x 100 beads 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

    • T3060 Monarch HMW DNA Extraction Kit for Tissue
    • T3000 Monarch Pestle Set
    • Monarch DNA 捕获珠
    • Monarch 捕获珠分离管
    • Monarch RBC 裂解液
    • T3056 Monarch gDNA Elution Buffer II
    • T3054 Monarch gDNA Nuclei Prep and Lysis Buffer Pack
    • T3061 Monarch HMW gDNA Tissue Lysis Buffer
    • T3062 Monarch Protein Separation Solution
    • T3055 Monarch Precipitation Enhancer
    • T3015 Monarch gDNA Wash Buffer

操作说明、说明书 & 用法

  • 操作说明

    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

  • 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。

    • manualT3050

  • 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 Cells & Blood (NEB #T3050) 
    • 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
    • Monarch® HMW DNA Extraction Kit improves sample preparation for Oxford Nanopore Technologies® sequencing of malaria parasites
    • 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 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?

  • 问题解决指南

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