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

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 (see figure "Highly Specific Cloning with a Shorter Ligation Time" and table "Time from PCR product to plated cells for different cloning methods"). Furthermore, as T is the most likely base to hybridize to non-complementary bases (i.e., G, C, and T; see references 1每4), 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 basepairing.

Highly Specific Cloning with a Shorter Ligation Time

 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, using the recommended ligation times for both protocols. Colony numbers were converted to relative percentages, with the QIAGEN PCR Cloningplus Kit procedure set at 100% for each comparison.  A  30 min ligation (QIAGEN PCR Cloningplus Kit recommendation).  B  4 h ligation (TA-based cloning recommendation).

 

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 "Robust QIAGEN EZ Competent Cells").

Robust QIAGEN EZ Competent Cells Do Not Require Recovery Incubation

QIAGEN EZ Competent Cells (>108 cfu/µg DNA), and 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.

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.

The QIAGEN PCR Cloning Kit procedure is much faster than topoisomerase-mediated, TA-based, and conventional sticky- and blunt-end cloning methods (see table "Time from PCR product to plated cells for different 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.

Time from PCR product to plated cells for different cloning methods

QIAGEN PCR Cloningplus Kit Topoisomerase-mediated cloning kit† TA-based cloning kit† Conventional ligase cloning
40 min ≡70 min ≡5.5 h ≡7.5 h

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.

Applications

QIAGEN PCR Cloning Kits are suitable for cloning of any PCR product that has a single A overhang at each 3' end. PCR products generated using Taq and other non-proofreading DNA polymerases can be directly cloned without any preparation.

QIAGEN also offers the QIAexpress UA Cloning Kit, for direct cloning of PCR products into the pQE-30 UA expression vector for high-level expression of 6xHis-tagged proteins. Follow the link for more information about the QIAexpress UA Cloning Kit.

紱釬忒聊: QIAGEN PCR Cloning Handbook 04/01 (PDF version, 132 KB)

QIAGEN PCR Cloning Kit

For direct cloning of PCR products generated by Taq and other non-proofreading DNA polymerases

Component Included Concentration
pDrive Cloning Vector 50 ng/µl
Ligation Master Mix 2x solution
Distilled water 每
QIAGEN EZ Competent Cells 每 每
SOC medium 每 每
Ordering Information
Product Contents Cat. No.
QIAGEN PCR Cloning Kit (10) For 10 reactions: 2x Ligation Master Mix (50 µl), pDrive Cloning Vector (0.5 µg), distilled water (1.7 ml) 231122
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) 231124

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QIAGEN PCR CloningPlus Kit

Includes QIAGEN EZ Competent Cells for added convenience

Component
Included
Concentration
pDrive Cloning Vector
50 ng/µl
Ligation Master Mix
2x solution
Distilled water
每
QIAGEN EZ Competent Cells
每
SOC medium
1x solution
Ordering Information
Product Contents Cat. No.
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
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