Qiagen, 231124, QIAGEN PCR Cloning Kit (40)

For 40 reactions: 2x Ligation Master Mix (200 µl), pDrive Cloning Vector (2.0 µg), Distilled water (1.7 ml)

Features

- Just 40 minutes from PCR product to plated cells
- Ready-to-use Ligation Master Mix
- High-specificity UA hybridization for efficient cloning
- Competent cells supplied with the QIAGEN PCR Cloning plus Kit
- Immediate plating of transformed competent cells

Product Details

QIAGEN PCR Cloning Kits provide 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. The QIAGEN PCR Cloning plus Kit also provides competent E. coli cells and SOC medium for efficient transformation.

Performance

QIAGEN PCR Cloning Kit: Topoisomerase-mediated cloning kit
40 min: ≥70 min

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 figure " Highly specific cloning with a shorter ligation time of 30 min " and table “Time from PCR product to plated cells for different cloning methods”).

Time from PCR product to plated cells for different cloning methods

The QIAGEN PCR Cloning plus Kit is supplied with QIAGEN EZ Competent Cells, which do not require this time-consuming recovery incubation to achieve high-efficiency transformation (>10 8 colony forming units (CFU) per microgram DNA; see figure " Transformation without recovery incubation ").

Principle

Component: QIAGEN PCR Cloning Kit
pDrive Cloning Vector: +
Ligation Master Mix: +
Distilled water: +
QIAGEN EZ Competent Cells: –
SOC medium: –

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 figure "Highly specific cloning with a shorter ligation time of 30 min"). 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.

The use of QIAGEN EZ Competent Cells in the QIAGEN PCR Cloning plus Kit makes the cloning procedure even faster and more convenient. Transformed cells are usually incubated in SOC medium to recover and to have time to express antibiotic resistance. In contrast, QIAGEN EZ Competent Cells do not require this time-consuming recovery incubation to achieve high-efficiency transformation (>10 8 colony forming units (CFU) per microgram DNA; see figure "Transformation without recovery incubation").

Components included with QIAGEN PCR Cloning Kits

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 EZ Competent Cells provided in the QIAGEN PCR Cloning plus Kit do not require a recovery incubation in SOC medium after transformation, and therefore can be plated immediately onto agar/ampicillin plates.

The QIAGEN PCR Cloning Kit procedure (see flowchart “ The QIAGEN PCR Cloning plus 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

QIAGEN PCR Cloning Kits are suitable for cloning 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 Kits.