标签归档：coli

克隆 E. coli 感受态细胞套装 |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

此为适用于高效转化的由四种大肠杆菌克隆菌株组成的试用套装。

重点

  • 1-3 x 109 cfu/μg pUC19 DNA 的转化效率
  • 抗 T1 噬菌体（fhuA2）
  • 含细胞复苏培养基和对照质粒
  • 无动物来源
  • 高性价比
  • 不收干冰费
产品类别:
Competent Cells Products,
Cloning Competent Cell Strains Products

应用:
Transformation

  • 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
    • C1010S     -80    
        NEB® 5-alpha Competent E. coli (High Efficiency) C2987HVIAL -80 2 x 0.05 ml Not Applicable
        NEB® 10-beta Competent E. coli (High Efficiency) C3019HVIAL -80 2 x 0.05 ml Not Applicable
        NEB® Stable Competent E. coli (High Efficiency) C3040HVIAL -80 2 x 0.05 ml Not Applicable
        NEB® Turbo Competent E. coli (High Efficiency) C2984HVIAL -80 2 x 0.05 ml Not Applicable
        SOC Outgrowth Medium B9020SVIAL 4 1 x 25 ml Not Applicable
        NEB® 10-beta/Stable Outgrowth Medium B9035SVIAL 4 1 x 25 ml Not Applicable
        pUC19 Vector N3041AVIAL -20 1 x 0.025 ml 50 pg/µl

  • 相关产品

    单独销售的组分

    • NEB® 5-alpha E. coli 感受态细胞（高效级）
    • NEB® 10-beta E. coli 感受态细胞（高效级）
    • NEB® Stable E. coli 感受态细胞（高效级）
    • NEB® Turbo E. coli 感受态细胞（高效级）
    • NEB® 10-beta/Stable Outgrowth 培养基
    • SOC Outgrowth 培养基

  • 注意事项

    1. 注意：本产品含危险物质 DMSO。处理前请先查看 MSDS。

    2. 贮存和处理：感受态细胞应在 -80℃ 条件下贮存。-20℃ 条件下贮存会显著降低转化效率。当细胞温度高于 -80℃ 时，即使不融化，细胞也会失去效率。

操作说明、说明书 & 用法

  • 使用指南

    • Chemical Transformation Tips
    • Competent E. coli Genome Sequences Tool

工具 & 资源

  • 选择指南

    • Competent Cell Product Comparison
    • Competent Cell Selection Guide

FAQs & 问题解决指南

  • FAQs

    1. Why did Synthetic Biologist Chris Voigt of MIT choose NEB 10-beta for DNA assembly and cloning?
    2. How should fragments be prepared for assembly using NEBuilder HiFi?

  • 问题解决指南

    • Troubleshooting Transformation Reactions

Express® E. coli Lysis Reagent  |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

NEBExpress® E. coli Lysis Reagent is a chemical lysis solution composed of a proprietary mix of non-ionic and zwitterionic detergents and Tris-based buffer. It allows disruption of E. coli cells without denaturing soluble proteins.

Figure 1: Optimal protein extraction with NEBExpress T4 Lysozyme in the presence of NEBExpress E. coli Lysis Reagent.

Express® E. coli Lysis Reagent  |

T7 Express E. coli  expressing vGFP was lysed with NEBExpress T4 Lysozyme (T4L) in 50 mM Tris-HCl pH 7.5 or in NEBExpress E. coli Lysis Reagent (NEB #P8116S): 4 UOD600 of cell pellet were resuspended in 200 µL of Tris buffer or NEBExpress E. coli Lysis Reagent and lysed for 5 minutes at room temperature in the presence of NEBExpress T4 Lysozyme at 0, 0.1 or 1 ug per 1 mL of cell suspension. The soluble proteins were harvested by centrifugation and analyzed on SDS-PAGE.

产品类别:
Competent Cells Products

应用:
High-throughput cloning and automation solutions,
Protein Expression in E. Coli,
Protein Expression

  • 产品组分信息

    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
    • P8116S     25    
        NEBExpress® E. coli Lysis Reagent P8116SVIAL 25 1 x 100 ml 1 X
    • P8116L     25    
        NEBExpress® E. coli Lysis Reagent P8116SVIAL 25 5 x 100 ml 1 X

  • 优势和特性

    Features

    • Scalable lysis reactions from small to large bacterial cell pellets and compatible with high throughput workflows
    • Suitable for extracting proteins, especially thermosensitive proteins vulnerable to mechanical lysis procedures
    • Fast and simple protocol: harvested cells are resuspended and briefly incubated in the Lysis Reagent to extract soluble proteins. Insoluble cell debris is removed by centrifugation, and the clarified extract is ready to use and compatible with affinity resins.
    • Compatible with analyses such as SDS-PAGE, Western blots, activity assays, immunoprecipitation, and downstream purification 
    • Suitable for disrupting most Gram-negative bacterial cells
    • Small (100 ml) and large kits (5 x 100 ml) are suitable for lysing 20 to 100 grams of cells, respectively.
     

  • 相关产品

    相关产品

    • m0303-dnase-i-rnase-free
    • T7 表达 lysY E. coli 感受态细胞（高效级）
    • BL21(DE3) E. coli 感受态细胞
    • T7 表达 E. coli 感受态细胞（高效级）
    • Amylose Resin
    • Amylose Resin High Flow
    • 几丁质树脂
    • NEBExpress® Ni-NTA 磁珠
    • NEBExpress® Ni Spin Columns
    • NEBExpress® Ni Resin
    • E8201 NEBExpress MBP 融合表达及纯化系统
    • IMPACT™ Kit
    • 亲水性链霉亲和素磁珠
    • SNAP-捕获磁珠
    • Color Prestained Protein Standard, Broad Range (10-250 kDa)
    • 蓝色蛋白上样染料
    • NEBExpress® T4 Lysozyme
    • 微球菌核酸酶

  • 注意事项

    1. NEBExpress E. coli Lysis Reagent is compatible with: 

      • Tris, phosphate, and PIPPS-based buffers ranging from pH 5.5-10.
      • EDTA. To prevent interference with metal affinity resins use < 0.1-0.5 mM EDTA. If using NEBExpress Ni Resin (NEB #S1428S) or Ni Spin Columns (NEB #S1427S/L), there is no limitation on EDTA use, as the resin is chemically resistant to EDTA chelation.
      • < 1 M NaCl (precipitation will occur at or above 1 M NaCl).
      • Protease inhibitors 
      • Reducing agent such as DTT, β-mercaptoethanol, TCEP, THP. To prevent interference with metal affinity resins, use 1-2 mM β-mercaptoethanol or 1-5 mM TCEP. NEBExpress Ni Resin (NEB #S1428) and Ni Spin Columns (NEB #S1427S/L) are both chemically resistant to these reducing agents. 
      • BCA Protein assay
      • SDS-PAGE and Western blots
      • 1 µL NEBExpress® T4 Lysozyme (NEB# P8115) per 1 mL of lysate for improved protein extraction efficiency. The lysate may become viscous due to the release of DNA. For optimal lysis yield, add 1mM CaCl2 and 1 µL of Micrococcal Nuclease (NEB #M0247) per 1 mL of lysate, mix and incubate for 5 minutes at room temperature.
       
    2. NEBExpress E. coli Lysis Reagent can be added directly to frozen cell pellets; however, allow this mixture to reach room temperature as the lysis will be more efficient.
    3. Use 0.025 – 0.075 mL NEBExpress E. coli Lysis Reagent per unit OD600 (UOD600) cells harvested. For example, for a 5 mL culture harvested at OD600 of 1, 5 UOD600 of cells are obtained (5 mL x 1 OD600 = 5 UOD600), thus requiring 0.125 to 0.375 mL of Lysis Reagent. 
    4. If weighing the cell pellet, use 5 mL NEBExpress E. coli Lysis Reagent per 1 gram of cell paste. Note that cells resuspended in Lysis Reagent at higher cell densities will not lyse as efficiently.
    5. Extraction efficiency is strain dependent. Efficiency is highest with BL21 strains, its derivatives with DE3 or lysY, E. coli K-12 strains, and various other Gram-negative bacteria (e.g., Salmonella, Vibrio, Pseudomonas).
    6. Storing NEBExpress E. coli Lysis Reagent at 4°C or below may cause precipitation of the detergents. However, the lysis efficiency of the reagent can be fully restored by incubation at room temperature with gentle mixing to dissolve any precipitate.

操作说明、说明书 & 用法

  • 操作说明

    1. Protocol for cell lysis using NEBExpress E. coli Lysis Reagent (NEB #P8116)

FAQs & 问题解决指南

  • FAQs

    1. How much NEBExpress® E. coli Lysis Reagent should I use?
    2. Why do I see precipitation in the NEBExpress® E. coli Lysis Reagent?
    3. Can I use NEBExpress® E. coli Lysis Reagent that has been frozen?
    4. Should I chill the NEBExpress® E. coli Lysis Reagent prior to use?
    5. Should I freeze the cell pellets prior to lysing with NEBExpress® E. coli Lysis Reagent?
    6. What buffer is present in the NEBExpress® E. coli Lysis Reagent?
    7. Can I supplement the NEBExpress® E. coli Lysis Reagent with NaCl?
    8. Can I change the pH of NEBExpress® E. coli Lysis Reagent?
    9. Is the NEBExpress® E. coli Lysis Reagent compatible with EDTA?
    10. Can I add a protease inhibitor to the NEBExpress® E. coli Lysis Reagent reaction?
    11. Do I need to add lysozyme to aid in lysis?
    12. My lysate is viscous; how can I resolve this?
    13. Can I use a reducing agent with NEBExpress® E. coli Lysis Reagent?
    14. Does NEBExpress® E. coli Lysis Reagent lyse gram-positive bacterial cells or yeast cells?
    15. Does NEBExpress® E. coli Lysis Reagent lyse gram-negative bacterial cells?
    16. My protein forms inclusion bodies. Can I still use this reagent to lyse my cells?
    17. Can I add NEBExpress® E. coli Lysis Reagent directly to my cell culture to lyse the cells?

  • 问题解决指南

    • Troubleshooting Guide for NEBExpress E. coli Lysis Reagent (NEB # P8116)

dam–/dcm– E. coli 感受态细胞 |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

缺失甲基转移酶的化学感受态大肠杆菌细胞，适合扩增无 Dam 和 Dcm 甲基化修饰的质粒。请注意，不建议将 dam– 菌株作为初级克隆/连接的宿主细胞。dam 突变会导致细胞内突变率增加和转化效率降低。DNA 应保持在 dam+ 菌株中，除非对无 Dam 或 Dcm 甲基化 DNA 有特定需求。

重点

  • 扩增无 Dam 和 Dcm 甲基化修饰的质粒
  • 无动物来源
  • 抗 T1 噬菌体（fhuA31）
  • 去除了非特异性核酸内切酶 I（endA1）的活性，以获得最高质量的质粒制备
  • K12 菌株

基因型

ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4 rfbD1 R(zgb210::Tn10) TetS endA1 rspL136 (StrR) dam13::Tn9 (CamR) xylA-5 mtl-1 thi-1 mcrB1 hsdR2

产品类别:
Cloning Competent Cell Strains Products

应用:
High-throughput cloning and automation solutions,
Transformation

  • 产品组分信息

    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
    • C2925H     -80    
        pUC19 Vector N3041AVIAL -20 1 x 0.025 ml 50 pg/µl
        SOC Outgrowth Medium B9020SVIAL 4 1 x 25 ml Not Applicable
        dam–/dcm– Competent E. coli C2925HVIAL -80 20 x 0.05 ml Not Applicable
    • C2925I     -80    
        pUC19 Vector N3041AVIAL -20 1 x 0.025 ml 50 pg/µl
        SOC Outgrowth Medium B9020SVIAL 4 1 x 25 ml Not Applicable
        dam–/dcm– Competent E. coli C2925IVIAL -80 6 x 0.2 ml Not Applicable

  • 特性和用法

    Antibiotic for Plasmid Selection

    用于质粒筛选的抗生素 使用浓度
    Ampicillin 100 µg/ml
    Carbenicillin 100 µg/ml
    Kanamycin 30 µg/ml
    Tetracycline 15 µg/ml

    货运单

    • Ships on dry ice

    抗生素耐药性

    • str
    • nit
    • cam

  • 优势和特性

    Features

    • 扩增无 Dam/Dcm 甲基转移酶的质粒

    应用特性

    热激时间对 dam–/dcm– E. coli 感受态细胞转化效率的影响：dam–/dcm– E. coli 感受态细胞 |
    按照所提供的高效级转化操作流程，用 100 pg pUC19 对照 DNA 转化 50 μl 感受态细胞，热激时间的变化范围为 0 至 80 秒。
    DNA 温育时间对 dam–/dcm– E. coli 感受态细胞转化效率的影响：dam–/dcm– E. coli 感受态细胞 |
    按照所提供的高效级转化操作流程，用 100 pg pUC19 对照 DNA 转化 50 μl 感受态细胞，DNA 温育时间的变化范围为 0 至 40 分钟。
    细胞复苏培养基对转化效率的影响：dam–/dcm– E. coli 感受态细胞 |
    按照所提供的高效级转化操作流程，用 100 pg pUC19 对照 DNA 转化 50 μl -/dcm– E.coli 感受态细胞，其中不同反应之间使用的细胞复苏培养基不同。NEB SOC Outgrowth 培养基的转化效率最高。

  • 相关产品

    相关产品

    • NEB® 10-beta E. coli 感受态细胞（高效级）
    • NEB® 5-alpha E. coli 感受态细胞（亚克隆效级）
    • NEB® 5-alpha F’ Iq E. coli 感受态细胞（高效级）
    • NEB® Turbo E. coli 感受态细胞（高效级）
    • NEB® 5-alpha E. coli 感受态细胞（高效级）
    • t1010-monarch-plasmid-miniprep-kit
    • Monarch® DNA 胶回收试剂盒
    • Monarch® PCR & DNA 纯化试剂盒（5 μg）

    单独销售的组分

    • SOC Outgrowth 培养基

  • 注意事项

    1. 注意：本产品含危险物质 DMSO。处理前请先查看 MSDS。
    2. 贮存和处理：感受态细胞应在 -80℃ 条件下贮存。-20℃ 条件下贮存会显著降低转化效率。当细胞温度高于 -80℃ 时，即使不融化，细胞也会失去效率。

操作说明、说明书 & 用法

  • 操作说明

    1. Transformation Protocol (C2925)

  • 使用指南

    • Additional E. coli Strain Genotypes
    • Chemical Transformation Tips
    • Competent E. coli Genome Sequences Tool
    • Genetic Markers
    • Making Unmethylated (Dam- Dcm-) DNA
    • McrA, McrBC and EcoKI Strain Phenotypes

  • 应用实例

    • Enhancing Transformation Efficiency

工具 & 资源

  • 选择指南

    • Characteristics of Select E.coli Strains
    • Competent Cell Product Comparison
    • Competent Cell Selection Guide

  • Web 工具

    • Competitor Cross-Reference Tool
    • NEBcloner®

FAQs & 问题解决指南

  • FAQs

    1. Can I transform unmethylated DNA into dam–/dcm– competent E.coli (C2925)?
    2. Does New England Biolabs offer a methyltransferase free strain of Competent E.coli?
    3. How long should I incubate cells on ice after DNA has been added (NEB #C2925H and NEB #C2925I)?
    4. Is dam–/dcm– (NEB #C2925H/C2925I) resistant to kanamycin?
    5. Is the DNA yield lower using dam–/dcm– strain (C2925)?
    6. What are the solutions/recipes (C2925)?
    7. What are the strain properties (C2925)?
    8. What is the difference between NEB #C2925H and NEB #C2925I?
    9. What is the optimal heat shock time for this strain (NEB #C2925H and NEB #C2925I)?
    10. Which strain of Competent E.coli should I use for general cloning?
    11. Why were the colonies at different size after transformation of dam–/dcm– competent E.coli (C2925)?
    12. How should I calculate the transformation efficiency (C2925)?
    13. Can I store competent cells at -20°C instead of -80°C?
    14. Which kind of transformation tubes should be used?
    15. What volume of DNA can be added into competent cells?
    16. What is the shelf life for this strain (NEB #C2925H and NEB #C2925I)?
    17. Are NEB’s competent cells compatible with the “Mix & Go” protocol?
    18. How should I store SOC Outgrowth Medium? The SOC I received with my competent cells recommends storage at either room temperature or 4°C, however, when I purchase it as a stand alone product, it recommends storing it at 4°C. Which is better?
    19. How should fragments be prepared for assembly using NEBuilder HiFi?

10-beta E. coli 电转感受态细胞 |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

NEB 10-beta E. coli 电转感受态细胞专为实现高效的电转化而优化。这些细胞非常适合 DNA 建库和所有克隆应用。

重点

  • DH10B™ 衍生菌株
  • 可克隆包括 BAC 和 cosmid 在内的大质粒
  • 可高效转化真核生物来源的甲基化 DNA，或来自 PCR、cDNA 和其它来源的非甲基化 DNA
  • 消除了非特异性核酸内切酶 I（endA1）的活性，以获得最高质量的质粒制备
  • 适用于蓝白斑筛选，通过 β-半乳糖苷酶基因（Φ80ΔlacZM15）的 α-互补实现
  • 减少克隆 DNA 的重组（recA）
  • 抗 T1 噬菌体（fhuA）
  • K-12 菌株
  • 无动物来源

基因型

Δ(ara-leu) 7697 araD139  fhuA ΔlacX74 galK16 galE15 e14-  Φ80dlacZΔM15  recA1 relA1 endA1 nupG  rpsL (StrR) rph spoT1 Δ(mrr-hsdRMS-mcrBC) 

产品类别:
Cloning Competent Cell Strains Products

应用:
Transformation

  • 产品组分信息

    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
    • C3020K     -80    
        NEB® 10-beta Electrocompetent E. coli C3020KVIAL -80 6 x 0.1 ml Not Applicable
        NEB® 10-beta/Stable Outgrowth Medium B9035SVIAL 4 1 x 25 ml Not Applicable
        pUC19 Vector N3041AVIAL -20 1 x 0.025 ml 50 pg/µl

  • 特性和用法

    Antibiotic for Plasmid Selection

    用于质粒筛选的抗生素 使用浓度
    Ampicillin 100 µg/ml
    Carbenicillin 100 µg/ml
    Chloramphenicol 33 µg/ml
    Kanamycin 30 µg/ml
    Tetracycline 15 µg/ml

    货运单

    • Ships on dry ice

    抗生素耐药性

    • str

  • 优势和特性

    Features

    电转技巧：

    1. 电转比色皿和微型离心管应在冰上预冷。
    2. 电转感受态细胞应该在冰上解冻，并通过小心轻弹管子使其悬浮。
    3. 将 DNA 加入到细胞后，就可以立即开始电转。无需将 DNA 与细胞一同温育。建议 DNA 溶液的最大体积为 2.5 µl。若添加大量 DNA，会降低转化效率。
    4. 盐和蛋白质等污染物可降低电转效率。理想情况下，用于转化的 DNA 应进行纯化并重悬于水或 TE 中。由于存在连接酶和盐，连接混合物的转化效率不到对照 pUC19 质粒的十分之一。如果直接使用，应将连接反应体系在 65℃ 条件下热失活 20 分钟，然后稀释 10 倍。为获得最佳结果，建议使用离心柱（NEB #T1030）纯化连接反应产物。
    5. 电穿孔条件受到比色皿和电穿孔仪的影响。如果使用的电穿孔仪或比色皿未在操作说明中指定，您可能需要优化电穿孔条件。建议使用 1 mm 间隙的比色皿（例如 BTX 610/613 型和 Bio-Rad #165-2089）。2 mm 间隙的比色皿需要更高的电压。
    6. 高浓度的盐或气泡可能会引起电弧。
    7. 在电穿孔后须立即向细胞中加入复苏培养基。延误一分钟会让效率降低为原来的三分之一。
    8. 冷而干燥的筛选平板会导致转化效率较低。将筛选平板在 37℃ 条件下预热 1 小时。使用 37℃ 预热的复苏培养基可让效率提高约 20%。
    9. 将未使用的细胞在干冰/乙醇浴中重新冷冻 5 分钟，然后在 -80℃ 条件下贮存。不要使用液氮。反复冻融会导致转化效率降低。

    应用特性

    10-beta E. coli 电转感受态细胞 |
    DNA 对转化效率和菌落产量的影响：在 DNA 起始量高达 10 ng 时电转效率仍然非常高，更高的 DNA 浓度则会降低转化效率。随着 puc19 DNA 起始量增加至 1 μg，总菌落数持续增加。

  • 相关产品

    相关产品

    • NEB® 10-beta E. coli 感受态细胞（高效级）
    • 电转连接酶®
    • m0269-phi29-dna-polymerase
    • t1010-monarch-plasmid-miniprep-kit
    • Monarch® DNA 胶回收试剂盒
    • Monarch® PCR & DNA 纯化试剂盒（5 μg）

    单独销售的组分

    • NEB® 10-beta/Stable Outgrowth 培养基

  • 注意事项

    1. 贮存和处理：感受态细胞应在 -80℃ 条件下贮存。-20℃ 条件下贮存会显著降低转化效率。当细胞温度高于 -80℃ 时，即使不融化，细胞也会失去效率。

操作说明、说明书 & 用法

  • 操作说明

    1. Electroporation Protocol (C3020)

  • 使用指南

    • Additional E. coli Strain Genotypes
    • Electroporation Tips
    • Genetic Markers
    • McrA, McrBC and EcoKI Strain Phenotypes
    • Traditional Cloning Quick Guide

工具 & 资源

  • 选择指南

    • Characteristics of Select E.coli Strains
    • Competent Cell Product Comparison

  • Web 工具

    • Competitor Cross-Reference Tool
    • NEBcloner®

FAQs & 问题解决指南

  • FAQs

    1. How should I calculate the Electrotransformation efficiency (C3020)?
    2. What are the strain properties (C3020)?
    3. What type of competent cells are suitable for transformation of DNA constructs created using Gibson Assembly?
    4. What type of competent cells are suitable for transformation of DNA constructs created using NEBuilder HiFi DNA Assembly Master Mix?
    5. How should I store SOC Outgrowth Medium? The SOC I received with my competent cells recommends storage at either room temperature or 4°C, however, when I purchase it as a stand alone product, it recommends storing it at 4°C. Which is better?
    6. How should I store the NEB 10-beta/Stable Outgrowth Medium?
    7. How should fragments be prepared for assembly using NEBuilder HiFi?