K0771 DNA末端修复 Thermo ,Fast DNA End Repair Kit/DNA末端快速修复试剂盒-试剂- bio-rad伯乐一级代理 |伯乐Aminex |伯乐电泳槽1658001| 伯乐ddPCR bio-rad授权代理商、biorad伯乐qpcr仪

K0771 DNA末端修复 Thermo ,Fast DNA End Repair Kit/DNA末端快速修复试剂盒

2024-10-24

Fast DNA End Repair Kit/DNA末端快速修复试剂盒

货号：K0771

规格：50 reactions

价格：2427

产品类型：DNA提取

品牌：Thermo Fisher

Thermo Scientific 快速 DNA 末端修复试剂盒可在 5 分钟内完成 DNA 末端的平末端化和磷酸化，以供后续在平末端连接中使用。试剂盒中的所有组分都包含预混试剂，从而减少移液步骤并提供便利。末端修复酶混合物含有经过优化的 T4 DNA 聚合酶和 Klenow 片段混合物，可实现对片段化 DNA 进行高效平末端化，并含有 T4 多聚核苷酸激酶 (PNK) 从而对 DNA 末端进行高效磷酸化。10X 末端修复反应混合物包含优化的反应缓冲液、ATP 和 dNTP。片段化基因组 DNA（限制性内切酶酶切、雾化或超声处理）、限制性内切酶酶切质粒 DNA、双链 cDNA 等样品和包含 dA 突出端的 PCR 产物均与此试剂盒兼容。特点在 DNA 末端修复反应中，片段化 DNA 被转化为含有 5'-磷酸基和 3'-羟基的平末端 DNA。末端修复酶混合物的 5'→3' 聚合酶活性填充 5' 突出 DNA 末端，而 3'→→5' 核酸外切酶活性去除 3'-突出端。T4 PNK 向非磷酸化 DNA 片段（如 PCR 产物）末端添加 5'-磷酸盐。（参见下面的表 1 和图 1）。应用• 双链 DNA（雾化 DNA、超声处理 DNA、限制性内切酶酶切 DNA、cDNA、PCR 产物）的平末端化和磷酸化包括• 末端修复酶混合物• 10X 末端修复反应混合物

优点：

-高效- 0.5 至 5 µg DNA 的平末端化和磷酸化-快速—反应可在 5 分钟内完成-便利—反应组分预先混合从而减少移液步骤

数据：

图1.Blunting and phosphorylation of different types of DNA ends

图2.End repair of different types of DNA endsOligoduplexes with different types of DNA ends (3’-, 5’-overhangs and blunt non-phosphorylated ends) were repaired using the Fast DNA End Repair Kit and kits from other vendors according to supplier’s recommendations in the presence of [γ-33P]-ATP. Reaction products were separated on denaturing polyacrylamide gel and phosphoimaged.Note: the two oligos of the oligoduplex migrate as two separate bands under a denaturating conditions. Non-specific end repair reaction products appear as additional bands of unexpected length.C1, C2 – synthetic radiolabeled oligoduplex identical to expected reaction productS1 – synthetic radiolabeled oligoduplexes identical to reaction substrate with 3’-overhangsS2 – synthetic radiolabeled oligoduplexes identical to reaction substrate with 5’-overhangs1 – oligoduplex end repaired with Fast DNA End Repair Kit (20°C, 5min)2 – oligoduplex end repaired with Fast DNA End Repair Kit (20°C, 20min)A – oligoduplex end repaired with Vendor A (20°C, 30min)B – oligoduplex end repaired with Vendor B (RT, 45min; 70°C, 10min)

图3.Efficient ligation after DNA end repairA 130bp PCR product with 3’- dA overhangs was end-repaired using the Fast DNA End Repair Kit and kits from other vendors according to supplier’s recommendations and subsequently ligated with T4 DNA Ligase (#EL0014). Ligation conditions: 200ng of 130bp PCR product ligated with 5U T4 DNA Ligase in 1X T4 DNA Ligase Buffer with 5%PEG 4000 for 1h at 22°C.M – GeneRuler 1 kb Plus DNA Ladder (#SM1333)C1 – PCR productC2 – PCR product after ligationT – PCR product after end repair using Fast DNA End Repair Kit (20°C, 5min) and ligationA – PCR product after end repair using Vendor A kit (20°C, 30min) and ligationB – PCR product after end repair using Vendor B kit (RT, 45min; 70°C, 10min) and ligation.

组成成分：

▪End Repair Enzyme Mix▪ 10X End Repair Reaction Mix

K0771 DNA末端修复 Thermo ,Fast DNA End Repair Kit/DNA末端快速修复试剂盒

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