QIAGEN PCR Cloning Kit
For direct cloning of PCR products generated by Taq DNA polymerases
The QIAGEN PCR Cloning Kit provides ready-to-use ligation reactions, which contain linearized cloning vectors that carry U overhangs at each 3' end, allowing PCR products containing 3'-end A overhangs to be directly ligated and cloned with high efficiency and speed. Buy Products
The QIAGEN PCR Cloning Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Product Details
Performance
The QIAGEN PCR Cloning Kit combines the latest ligation technology with a unique combination of time-saving features for fast, easy, and highly efficient cloning of PCR products generated using Taq and other nonproofreading DNA polymerases. The QIAGEN PCR Cloning Kit outperformed kits tested from other suppliers, ensuring successful results. Cloning into the pDrive Cloning Vector is much faster compared to TA-based cloning vectors (see figures "Highly specific cloning with a shorter ligation time of 30 min" and "Highly specific cloning with a ligation time of 4 h", and table).
Principle
The pDrive Cloning Vector (see figure "pDrive Cloning Vector") provides highly efficient cloning of PCR products through UA hybridization. The vector is supplied in a linear form with a U overhang at each 3' end, which hybridizes with high specificity to the A overhang of PCR products generated by Taq and other nonproofreading DNA polymerases. The pDrive Cloning Vector (see figure "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. PCR products are efficiently cloned into the pDrive Cloning Vector in less time than is required for TA-based cloning vectors (see figures "Highly specific cloning with a shorter ligation time of 30 min" and "Highly specific cloning with a ligation time of 4 h"). Furthermore, as T is the most likely base to hybridize to noncomplementary bases (i.e., G, C, and T), vectors with a T overhang are more likely to self-anneal or to clone primers or annealed primers, leading to an increased number of false-positive colonies. In contrast, the higher cloning efficiency of the pDrive Cloning Vector indicates that U has a lower tolerance for nonspecific base pairing. The Ligation Master Mix, supplied in a convenient premixed format, is specifically designed to provide optimal hybridization conditions for efficient cloning.
Procedure
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. The QIAGEN PCR Cloning Kit procedure is much faster than topoisomerase-mediated, TA-based, and conventional sticky- and blunt-end cloning methods. Ligation takes 30 minutes and transformation and plating using QIAGEN EZ Competent Cells takes only 10 minutes, making the complete procedure — from PCR product to plated cells — just 40 minutes.
Applications
The QIAGEN PCR Cloning Kit is suitable for cloning of any PCR product that has a single A overhang at each 3' end. PCR products generated using Taq and other nonproofreading DNA polymerases can be directly cloned without any preparation. Kits offering proofreading DNA polymerases, such as the HotStar HiFidelity Polymerase Kit and the QIAGEN LongRange PCR Kit, generate PCR products with A overhangs, and are also highly suited for direct use with the QIAGEN PCR Cloning Kit.
Specifications
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