直接克隆PCR产物

  • 从PCR产物到涂板只需40分钟
  • 即用型Ligation Master Mix
  • 高特异性UA杂交,用于高效克隆
  • 试剂盒提供感受态细胞
  • 转化细胞可立刻涂板
QIAGEN PCR Cloningplus Kit提供即用型连接反应,包括3'端含有U突出末端的线性克隆载体,3'端带有A突出末端的PCR产物可直接进行高效、快速的连接和克隆。QIAGEN PCR Cloningplus Kit还提供E. coli感受态细胞和SOC培养基,用于高效转化。
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QIAGEN PCR Cloningplus Kit (10)
For 10 reactions: 2x Ligation Master Mix (50 µl), pDrive Cloning Vector (0.5 µg), Distilled water (1.7 ml), QIAGEN EZ Competent Cells (10 tubes, 50 µl each), SOC medium (2 x 1.9 ml)
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QIAGEN PCR Cloningplus Kit (40)
For 40 reactions: 2x Ligation Master Mix (200 µl), pDrive Cloning Vector (2.0 µg), Distilled water (1.7 ml), QIAGEN EZ Competent Cells (40 tubes, 50 µl each), SOC medium (6 x 1.9 ml)
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The QIAGEN PCR Cloningplus Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Highly specific cloning with a shorter ligation time of 30 min.|Transformation without recovery incubation.|Highly specific cloning with a ligation time of 4 h.|The QIAGEN PCR Cloning plus Kit procedure.|pDrive Cloning Vector.|
The effect of ligation time on cloning efficiency was compared for the QIAGEN PCR Cloningplus Kit and a TA-based cloning kit (Supplier I) using PCR products of different length (0.5 kb, 1 kb, and 3 kb). The recommended protocol for each kit was followed. Colony numbers were converted to relative percentages, with the QIAGEN PCR Cloningplus Kit procedure set at 100% for each comparison. The ligation was performed for 30 min (QIAGEN PCR Cloningplus Kit recommendation).|QIAGEN EZ Competent Cells do not require recovery incubation. QIAGEN EZ Competent Cells (>108 cfu/µg DNA), TOP 10F (Supplier I; >109 cfu/µg DNA), and JM109 (Supplier P; >108 cfu/µg DNA) competent cells were transformed with pUC18 plasmid DNA. The recommended protocol from each supplier was followed, except that all cells were plated immediately onto agar/ampicillin plates without a recovery incubation in SOC medium. Colony numbers were converted to relative percentages, with QIAGEN EZ Competent Cells set at 100%. Colony numbers were not normalized for transformation efficiency. Normalization would result in an even higher transformation efficiency for QIAGEN EZ Competent Cells.|The effect of ligation time on cloning efficiency was compared for the QIAGEN PCR Cloningplus Kit and a TA-based cloning kit (Supplier I) using PCR products of different length (0.5 kb, 1 kb, and 3 kb). The recommended protocol for each kit was followed. Colony numbers were converted to relative percentages, with the QIAGEN PCR Cloningplus Kit procedure set at 100% for each comparison. The ligation was performed for 4 hours (TA-based cloning recommendation).|Simply mix the PCR product directly with pDrive Cloning Vector and Ligation Master Mix, incubate, and then add the ligation reaction to competent cells for transformation. QIAGEN EZ Competent Cells do not require a recovery incubation in SOC medium after transformation, and therefore can be plated immediately onto agar/ampicillin plates.|The pDrive Cloning Vector has a number of useful features designed to facilitate analysis of cloned PCR products. These include a large number of unique restriction enzyme recognition sites, universal sequencing primer sites, and promoters for in vitro transcription. In addition, the vector allows both ampicillin and kanamycin selection as well as blue/white screening of recombinant colonies.|
Performance

QIAGEN PCR Cloningplus Kit整合了最新的具有省时特点的连接技术,可对Taq和其他非高保真DNA聚合酶产生的PCR产物进行快速、简单、高效的克隆。QIAGEN PCR Cloningplus Kit优于其他供应商的试剂盒,确保结果成功。与基于TA的克隆载体相比,使用pDrive Cloning Vector克隆更加快速(参见"Highly specific cloning with a shorter ligation time of 30 min"、"Highly specific cloning with a ligation time of 4 h"和表格)。QIAGEN PCR Cloningplus Kit提供QIAGEN EZ Competent Cells,无需费时的复苏过程即可高效转化(每微克DNA >108个菌落形成单位(CFU);参见"Transformation without recovery incubation")。

从PCR产物到涂板不同的克隆方法所需的时间
QIAGEN PCR Cloningplus Kit Topoisomerase介导的克隆试剂盒 基于TA的克隆试剂盒 常规连接酶克隆
40 min ≥70 min ≥5.5 h ≥7.5 h
Principle

pDrive Cloning Vector(参见"pDrive Cloning Vector")通过UA杂交可对PCR产物进行高效克隆。载体是3'端带有U突出末端的线性形式,能与Taq和其他非高保真DNA聚合酶产生的带有A突出末端的PCR产物进行高特异性杂交。pDrive Cloning Vector具有很多有用的特征,便于对克隆的PCR产物进行分析。这些特征包括多种独特的限制性酶切识别位点、通用测序引物位点和用于体外转录的启动子。此外,载体还可进行氨苄青霉素和卡那霉素筛选,以及对重组克隆的蓝/白斑筛选。

PCR产物可高效克隆到pDrive Cloning Vector,与基于TA的克隆载体相比,所需时间更少。而且,T是最可能与非互补碱基(如G、C和T)杂交的碱基,带有T突出末端的载体更可能发生自连或连接克隆引物或退火引物,导致假阳性克隆数目增加。相比起来,pDrive Cloning Vector具有更高克隆效率,表明U对非特异性碱基配对有更低容忍度。

Ligation Master Mix采用预混形式,专用于为高效克隆提供最佳的杂交环境。

使用QIAGEN EZ Competent Cells能让克隆步骤更加快速和方便。转化的细胞通常都需要在SOC培养基中孵育复苏,需要时间来表达抗性。与此不同,QIAGEN EZ Competent Cells不需要这个耗时的复苏孵育步骤即可高效转化(每微克DNA >108个菌落形成单位(CFU)。

QIAGEN PCR Cloningplus Kit的组成
成分 包括 浓度
pDrive Cloning Vector
50 ng/µl
Ligation Master Mix
2x solution
Distilled Water
QIAGEN EZ Component Cells
SOC medium
1x solution
Procedure
直接将PCR产物和pDrive Cloning Vector及Ligation Master Mix简单混合,再将连接反应液加入到感受态细胞中进行转化。QIAGEN EZ Competent Cells在转化后无需在SOC培养基复苏孵育,可直接在琼脂/氨苄青霉素培养基上涂板。

与拓扑异构酶法、TA法、常规粘性末端和平末端克隆方法相比,QIAGEN PCR Cloningplus Kit(参见"PCR Cloning Kit procedure")操作流程更加快速。连接需要30分钟,使用QIAGEN EZ Competent Cells进行转化和涂板只需10分钟,从PCR产物到涂板的整个操作流程只需40分钟。

Applications
QIAGEN PCR Cloningplus Kit适用于克隆任何3'端带有单独A突出末端的PCR产物。使用Taq和其他非高保真DNA聚合酶产生的PCR产物无需任何制备,可直接进行克隆。提供高保真DNA聚合酶的试剂盒,如HotStar HiFidelity Polymerase Kit和QIAGEN LongRange PCR Kit,可产生带有A突出末端的PCR产物,非常适合与QIAGEN PCR Cloningplus Kit配合使用。
Feature
Specifications
Applications Cloning of A overhang PCR products
Competent cells QIAGEN EZ Competent Cells
Overhang U overhang
Reaction type UA hybridization
Vector included pDrive Cloning Vector

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