标签归档：dna

Quick Blunting™ Kit | NEB酶试剂 New England Biolabs

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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。

Quick Blunting™ Kit

The Quick Blunting™ Kit is used to convert DNA with incompatible 5´or 3´overhangs to 5´phosphorylated, blunt-ended DNA for efficient blunt-end ligation into DNA cloning vectors.

  • Restriction enzyme digested DNA is blunted in less than 30 minutes
  • Reactions are performed at room temperature in a ready-to-use mix
  • Suitable for restriction enzyme digested DNA, sheared or nebulized DNA or PCR product

精选视频

Quick Blunting™ Kit | NEB酶试剂 New England Biolabs

DNA Blunting Tutorial

观看其他视频

库存
货号 浓度 规格 目录价 北京 上海 广州 成都 苏州
E1201S 不适用 20 reactions ¥969.00 有 有 无 有 有
E1201L 不适用 100 reactions ¥3,909.00 有 无 无 无 无
如何订购
Please enter a quantity for at least one size

Quick Blunting™ Kit | NEB酶试剂 New England Biolabs Need a custom/large volume order? Contact Us

Bulk packaging may also be available and requested for large recurring orders.
Learn More

产品信息

The Quick Blunting Kit is used to convert DNA with incompatible 5´ or 3´ overhangs to 5´ phosphorylated, blunt-ended DNA for efficient blunt-end ligation into DNA cloning vectors. DNA is blunted using T4 DNA Polymerase (NEB #M0203) which has both 3´ → 5´ exonuclease activity and 5´ → 3´ polymerase activity. T4 Polynucleotide Kinase (NEB #M0201) is included in the enzyme mix for phosphorylation of the 5´ ends of blunt-ended DNA for subsequent ligation into a cloning vector. This kit is optimized for blunting up to 5 µg of DNA in a single reaction. To learn more about how to identify what type of overhang you have, visit this video tutorial.

Blunt Enzyme Mix supplied in: 100 mM KCl, 10 mM Tris-HCl(pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% Triton X-100 and 50% Glycerol.

1X Blunting Buffer:
100 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
0.025% Triton X-100
5 mM dithiothreitol
pH 7.5 at 25°C

产品类别:
DNA Manipulation Products

应用:
Blunting,
Phosphorylation (Kinase),
PCR,

Fast Cloning: Accelerate your cloning workflows with reagents from NEB

  • 试剂盒组成

    Kit 组分

    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
    • E1201S     -20    
        Blunting Enzyme Mix M1201AVIAL -20 1 x 0.025 ml 不适用
        10X Blunting Buffer B1201SVIAL -20 1 x 0.5 ml 10 X
        Deoxynucleotide Solution Mix (1 mM) N1201AVIAL -20 1 x 0.1 ml 1 mM
    • E1201L     -20    
        Blunting Enzyme Mix M1201AAVIAL -20 1 x 0.125 ml 不适用
        10X Blunting Buffer B1201SVIAL -20 1 x 0.5 ml 10 X
        Deoxynucleotide Solution Mix (1 mM) N1201AAVIAL -20 1 x 0.5 ml 1 mM

  • 特性和用法

  • 优势和特性

    应用特性

    Prepare sheared, nebulized or restriction enzyme digested DNA for blunt-ended ligation into a plasmid, cosmid, fosmid or BAC vector

    Prepare PCR products for efficient blunt-end cloning

  • 相关产品

    相关产品

    • Quick Blunting™ and Quick Ligation™ Kits
    • Quick T4 DNA Ligase
    • Monarch® Plasmid Miniprep Kit 
    • Monarch® DNA Gel Extraction Kit
    • Monarch® PCR & DNA Cleanup Kit (5 μg)

  • 注意事项
    1. Restriction enzyme digested DNA can be blunted directly without purification. The Blunt Enzyme Mix has been optimized in Blunting Buffer, but is also active in NEBuffers 1,2,3 and 4, as well as BamHI, EcoRI and DpnII unique buffers when supplemented with dNTPs and dithiothreitol. There is a small reduction in ligation fidelity in these buffers. Transformation efficiency is lowest in NEBuffer 1 where the total yield is about 50% of optimum.
    2. ATP is not necessary for T4 Polynucleotide Kinase activity in the kit. The dATP and dTTP in the dNTP mix act as phosphate donors.
    3. The blunted reaction must be purified prior to phosphatase treatment by using a commercial purification kit, phenol extraction/ethanol precipitation, or gel electrophoresis.
    4. PCR generated DNA must be purified before blunting by using a commercial purification kit, such as Monarch® PCR & DNA Cleanup Kit (NEB #T1030), phenol extraction/ethanol precipitation, or gel electrophoresis.

操作说明、说明书 & 用法

  • 操作说明
    1. Blunting protocol for NEB PCR Cloning Kit
    2. Protocol for the Quick Blunting Kit (E1201)
    3. Transformation Protocol
    4. Quick Ligation Protocol (M2200)

工具 & 资源

  • 选择指南
    • Blunting Selection Chart

  • Web 工具
    • NEBcloner®

FAQs & 问题解决指南

  • FAQs
    1. Which ligase is recommended to ligate DNA treated with the Quick Blunting Kit?
    2. Can I use regular ligase to ligate my blunted product?
    3. I’ve blunted my DNA and now need to dephosphorylate the reaction with Antarctic Phosphatase. Do I need to purify the reaction?
    4. Does the PCR product need to be purified before blunting with the Quick Blunting Kit? What methods can be used to purify the product?
    5. I’ve digested my DNA and now want to blunt directly without purifying, can I add the blunting reagents directly to the restriction digest?
    6. I’ve blunted my sonicated gDNA for 15 minutes instead of the recommended 30 minute incubation time, will the reaction still work?
    7. I’ve accidentally skipped the heat-kill step after the blunting reaction. Will the ligation still work?

  • 问题解决指南
    • EpiMark® Methylated DNA Enrichment Kit Troubleshooting Guide

引用 & 技术文献

  • 引用文献

    产品引用文献查找工具

    Quick Blunting™ Kit | NEB酶试剂 New England Biolabs Powered by Bioz See more details on Bioz

质控、安全 & 法规

  • 质控分析
    每一新批次的 NEB 产品都要经过质控，以满足指定的质量标准，对特定产品的质量标准和每一批次的检测数据可以通过产品说明表格、COA、产品信息卡或者产品手册进行查阅或下载。关于 NEB 产品质控的详细信息，可从此处查阅。

  • 产品说明与变更通知

    产品说明与变更通知

    产品说明表包含产品的储存温度、有效期和规格，这些文件的命名规则如下：[货号]_[规格]_[版本]

    • E1201S_L_v1

  • CoA
    CoA 文件包含单个批次产品的储存温度、有效期和质控，这些文件的命名规则如下： [货号]_[规格]_[版本]_[Lot#]

    • E1201S_L_v1_0101801
    • E1201L_v1_10013484
    • E1201L_v1_10013485
    • E1201L_v1_10038381
    • E1201S_v1_10038380
    • E1201S_v1_10053797
    • E1201S_v1_10058037
    • E1201S_v1_10062856
    • E1201L_v1_10053799
    • E1201L_v1_10062855
    • E1201S_v1_10080108
    • E1201L_v1_10085502
    • E1201L_v1_10102455
    • E1201S_v1_10107427
    • E1201S_v1_10142224
    • E1201L_v1_10149861
    • E1201S_v1_10163391

  • SDS
    以下 SDS 文件可以帮助您安全地使用该产品

    • Blunting Enzyme Mix
    • 10X Blunting Buffer
    • Deoxynucleotide Solution Mix (1 mM)

DNA Polymerase I, Large (Klenow) Fragment |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。

DNA Polymerase I, Large (Klenow) Fragment

DNA Polymerase I, Large (Klenow) fragment was originally derived as a proteolytic product of E.coli DNA polymerase that retains polymerase and 3’ —> 5’ exonuclease activity

  • Removal of 3’ overhangs or fill-in of 5’ overhangs to form blunt ends
  • Lacks 5’ —> 3’ exonuclease activity
  • Generates probes using random primers
  • Second strand cDNA synthesis

精选视频

DNA Polymerase I, Large (Klenow) Fragment |

DNA Blunting Tutorial

观看其他视频

库存
货号 浓度 规格 目录价 北京 上海 广州 成都 苏州
M0210V 5,000 units/ml 100 units ¥359.00 有 有 无 无 有
M0210S 5,000 units/ml 200 units ¥679.00 有 有 有 有 有
M0210L 5,000 units/ml 1,000 units ¥2,699.00 有 有 有 无 有
M0210M 50,000 units/ml 1,000 units ¥2,699.00 有 有 有 无 无
如何订购
Please enter a quantity for at least one size

DNA Polymerase I, Large (Klenow) Fragment | Need a custom/large volume order? Contact Us

Bulk packaging may also be available and requested for large recurring orders.
Learn More

产品信息

DNA Polymerase I, Large (Klenow) Fragment is a proteolytic product of E. coli DNA Polymerase I which retains polymerization and 3’→ 5′ exonuclease activity, but has lost 5’→ 3′ exonuclease activity (1). Klenow retains the polymerization fidelity of the holoenzyme without degrading 5′ termini.

重点

 

产品来源

An E. coli strain that contains the E. coli polA gene that has had its 5’→3′ exonuclease domain removed.

产品类别:
DNA Manipulation Products

应用:
Blunting,
DNA Sequencing,
Polymerases for DNA Manipulation,

RT-PCR & cDNA Synthesis,
RT-qPCR, RT-PCR and cDNA Synthesis,

PCR

  • 产品组分信息

    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
    • M0210V     -20    
        DNA Polymerase I, Large (Klenow) Fragment M0210VVIAL -20 1 x 0.02 ml 5,000 units/ml
        NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
    • M0210S     -20    
        DNA Polymerase I, Large (Klenow) Fragment M0210SVIAL -20 1 x 0.04 ml 5,000 units/ml
        NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
    • M0210L     -20    
        DNA Polymerase I, Large (Klenow) Fragment M0210LVIAL -20 1 x 0.2 ml 5,000 units/ml
        NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
    • M0210M     -20    
        DNA Polymerase I, Large (Klenow) Fragment M0210MVIAL -20 1 x 0.02 ml 50,000 units/ml
        NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X

  • 特性和用法

    单位定义

    One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 37°C.

    反应条件

    1X NEBuffer™ 2
    Incubate at 25°C

    1X NEBuffer™ 2
    50 mM NaCl
    10 mM Tris-HCl
    10 mM MgCl2
    1 mM DTT
    (pH 7.9 @ 25°C)

    贮存溶液

    25 mM Tris-HCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    pH 7.4 @ 25°C

    热失活

    75°C for 20 min

    分子量

    理论上的: 68000 daltons

    5′ – 3′ 核酸外切酶

    No

    3′ – 5′ 核酸外切酶

    Yes

    链置换

    +

    单位活性检测条件

    1X NEBuffer 2, 33 μM dNTPs including [3H]-dTTP and 70 μg/ml denatured herring sperm DNA.

    错配率

    ~ 18×10-6bases

  • 优势和特性

    应用特性

    • DNA sequencing by the Sanger dideoxy method (2)
    • Fill-in of 5´ overhangs to form blunt ends (3)
    • Removal of 3´ overhangs to form blunt ends (3)
    • Second strand cDNA synthesis
    • Second strand synthesis in mutagenesis protocols (4)

  • 相关产品

    相关产品

    • Deoxynucleotide (dNTP) Solution Set
    • Deoxynucleotide (dNTP) Solution Mix
    • Monarch® Plasmid Miniprep Kit 
    • Monarch® DNA Gel Extraction Kit
    • Monarch® PCR & DNA Cleanup Kit (5 μg)

    单独销售的组分

    • NEBuffer 2

  • 注意事项
    1. Protocol for blunting ends by 3′ overhang removal and 3′ recessed end fill-in: 
      • DNA should be dissolved in 1X NEBuffer 1-4 or T4 DNA Ligase Reaction Buffer supplemented with 33 μM each dNTP. Add 1 unit DNA Polymerase I, Large (Klenow) Fragment per microgram DNA and incubate 15 minutes at 25°C. Stop reaction by adding EDTA to a final concentration of 10 mM and heating at 75°C for 20 minutes. 
      • CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times may result in recessed ends due to the 3’→ 5′ exonuclease activity of the enzyme.
    2. When DNA Polymerase I, Large (Klenow) Fragment is used to sequence DNA using the dideoxy method of Sanger et al., 1 unit/5 μl reaction volume is recommended.
    3. DNA Polymerase I, Large (Klenow) Fragment is also active in all four NEBuffers and T4 DNA Ligase Reaction Buffer when supplemented with dNTPs.

  • 参考文献
    1. Jacobsen, H., Klenow, H. and Overgaard-Hansen, K. (1974). Eur. J. Biochem.. 45, 623-627.
    2. Sanger, F. et al. (1977). Proc. Natl. Acad. Sci. USA. 74, 5463-5467.
    3. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.40-5.43. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
    4. Gubler, U. (1987). In S.L. Berger and A.R. Kimmel(Ed.), Methods in Enzymology. 152, 330-335. San Diego: Academic Press.
    5. Bebenek, K., Joyce, C.M., Fitzgerald, M.P. and Kunkel, T.A. (1990). J. Bio. Chem . 265, 13878-13887.

操作说明、说明书 & 用法

  • 操作说明
    1. Protocol for blunting ends by 3′ overhang removal and fill-in of 3′ recessed (5′ overhang) ends using DNA Polymerase I, Large (Klenow) Fragment (M0210)

  • 使用指南
    • Activity of DNA Modifying Enzymes in rCutSmart™ Buffer

工具 & 资源

  • 选择指南
    • Blunting Selection Chart
    • DNA Polymerase Selection Chart

  • Web 工具
    • NEBcloner®

FAQs & 问题解决指南

  • FAQs
    1. Can DNA Polymerase I, Large (Klenow) Fragment be used in other NEBuffers, including rCutSmart?
    2. Can DNA Polymerase I, Large (Klenow) Fragment be used to blunt DNA?
    3. Can DNA Polymerase I, Large (Klenow) Fragment be used to fill in 3′ overhangs?
    4. Can DNA Polymerase I, Large (Klenow) Fragment be used to chew back 5′ overhangs?
    5. Can DNA Polymerase I, Large (Klenow) Fragment be heat inactivated?
    6. Are the nucleotides needed to remove a 3′ overhang with DNA Polymerase I, Large (Klenow) Fragment?
    7. What are the main causes for blunting reaction failure using DNA Polymerase I, Large (Klenow) Fragment?
    8. Can DNA Polymerase I, Large (Klenow) Fragment be used in labeling reactions and partial fill in reactions?
    9. Is DNA Polymerase I, Large (Klenow) Fragment the enzyme of choice for chewing back 3′ overhangs and filling in 5′ overhangs (3′ recessed ends)?
    10. Are NEB DNA Polymerases supplied with dNTPs?

  • 实验技巧
    Excess enzyme, temperatures above 25°C, or limited dNTP concentrations can cause excessive 3’ ? 5’ exonuclease degradation, which eliminates blunt end formation.

引用 & 技术文献

  • 引用文献

    产品引用文献查找工具

    DNA Polymerase I, Large (Klenow) Fragment | Powered by Bioz See more details on Bioz

质控、安全 & 法规

  • 质控分析
    每一新批次的 NEB 产品都要经过质控，以满足指定的质量标准，对特定产品的质量标准和每一批次的检测数据可以通过产品说明表格、COA、产品信息卡或者产品手册进行查阅或下载。关于 NEB 产品质控的详细信息，可从此处查阅。

  • 产品说明与变更通知

    产品说明与变更通知

    产品说明表包含产品的储存温度、有效期和规格，这些文件的命名规则如下：[货号]_[规格]_[版本]

    • M0210M_v1
    • M0210S_L_v1

  • CoA
    CoA 文件包含单个批次产品的储存温度、有效期和质控，这些文件的命名规则如下： [货号]_[规格]_[版本]_[Lot#]

    • M0210M_v1_0891509
    • M0210S_L_v1_0891509
    • M0210S_L_v1_0891512
    • M0210S_L_v1_0891603
    • M0210M_v1_0891603
    • M0210M_v1_0891608
    • M0210S_L_v1_0891608
    • M0210M_v1_0891703
    • M0210S_L_v1_0891703
    • M0210M_v1_0891709
    • M0210S_L_v1_0891709
    • M0210M_v1_0891803
    • M0210S_L_v1_0891803
    • M0210L_v1_10007540
    • M0210L_v1_10025743
    • M0210S_v1_10024368
    • M0210L_v1_10028508
    • M0210M_v1_10028233
    • M0210S_v1_10028423
    • M0210L_v1_10034214
    • M0210M_v1_10034564
    • M0210S_v1_10034001
    • M0210L_v1_10036837
    • M0210S_v1_10037080
    • M0210L_v0_10041498
    • M0210L_v1_10046211
    • M0210S_v1_10043965
    • M0210S_v1_10050337
    • M0210L_v1_10053482
    • M0210S_v1_10055962
    • M0210L_v1_10057607
    • M0210L_v1_10058269
    • M0210S_v1_10058172
    • M0210L_v1_10062077
    • M0210M_v1_10057608
    • M0210S_v1_10062081
    • M0210L_v1_10065299
    • M0210S_v1_10066373
    • M0210L_v1_10075466
    • M0210S_v1_10075464
    • M0210S_v1_10079480
    • M0210M_v1_10087408
    • M0210L_v1_10088146
    • M0210S_v1_10087380
    • M0210S_v1_10094981
    • M0210L_v1_10100634
    • M0210M_v1_10101977
    • M0210L_v1_10106852
    • M0210S_v1_10107645
    • M0210S_v1_10113999
    • M0210L_v1_10117205
    • M0210M_v1_10117349
    • M0210M_v1_10123077
    • M0210S_v1_10127909
    • M0210L_v1_10136292
    • M0210M_v1_10137266
    • M0210L_v1_10149552
    • M0210S_v1_10150626
    • M0210L_v1_10155130
    • M0210M_v1_10154966
    • M0210L_v1_10162372

  • SDS
    以下 SDS 文件可以帮助您安全地使用该产品

    • DNA Polymerase I, Large (Klenow) Fragment
    • NEBuffer™ 2