For direct cloning of PCR products
  • 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 kit
  • Immediate plating of transformed competent cells
The QIAGEN PCR Cloningplus 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. The QIAGEN PCR Cloningplus Kit also provides competent E. coli cells and SOC medium for efficient transformation.
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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)
231222
$269.00
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)
231224
$954.00
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

The QIAGEN PCR Cloningplus 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 Cloningplus Kit outperformed kits tested from other suppliers, ensuring successful results. Cloning into the pDrive Cloning Vector is much faster compared with 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). The QIAGEN PCR Cloningplus Kit is supplied with QIAGEN EZ Competent Cells, which do not require this time-consuming recovery incubation to achieve high-efficiency transformation (>108 colony forming units (CFU) per microgram DNA; see figure "Transformation without recovery incubation").

Time from PCR product to plated cells for different cloning methods
QIAGEN PCR Cloningplus Kit Topoisomerase-mediated cloning kitTA-based cloning kit Conventional ligase cloning
40 min ≥70 min ≥5.5 h ≥7.5 h

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 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 tme 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.

The use of QIAGEN EZ Competent Cells 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 (>108 colony forming units (CFU) per microgram DNA; see figure "Transformation without recovery incubation").

Components included with the QIAGEN PCR Cloningplus Kit
Component IncludedConcentration
pDrive Cloning Vector
50 ng/µl
Ligation Master Mix
2x solution
Distilled Water
QIAGEN EZ Component Cells
SOC medium
1x solution

 

 

 

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. 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 QIAGEN PCR Cloningplus Kit procedure (see flowchart "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 Cloningplus 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 Cloningplus Kit.
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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