Cloning – QIAGEN^® PCR Cloning System

High-specificity UA hybridization provided by new QIAGEN® PCR Cloning Kits and robust QIAGEN EZ Competent Cells allow fast and efficient cloning of PCR products generated using Taq and other non-proofreading DNA polymerases.

Features and benefits

• Just 40 minutes from PCR product to plated cells
• Ready-to-use Ligation Master Mix and immediate plating of transformed QIAGEN EZ Competent Cells
• High-specificity UA hybridization for highly efficient cloning
• Choice of formats — with or without competent cells

Principle

The 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 non-proofreading DNA polymerases. The Ligation Master Mix, supplied in a convenient premixed format, is specifically designed to provide optimal hybridization conditions for efficient cloning.

PCR products are efficiently cloned into the pDrive Cloning Vector in less time than is required for TA-based cloning vectors (Figure 1). Furthermore, as T is the most likely base to hybridize to non-complementary bases ( i.e., G, C, and T; see references 1–4), vectors with a T overhang are more likely to self-anneal or to clone primers, annealed primers, or incomplete PCR products, 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 basepairing.

Procedure

Simply mix your 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.

Table 1. Time from PCR product to plated cells for different cloning methods

The QIAGEN PCR Cloning Kit procedure is much faster than topoisomerase-mediated, TA-based, and conventional sticky- and blunt-end cloning methods (Table 1). 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.

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.

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