QIAGEN PCR Cloning Kits

For direct cloning of PCR products generated by Taq DNA polymerases

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QIAGEN PCR Cloning Kit (40)

Cat. No. / ID: 231124

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

QIAGEN PCR Cloning Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

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

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

QIAGEN PCR Cloning Kit	Topoisomerase-mediated cloning kit	TA-based cloning kit	Conventional ligase cloning
40 min	≥70 min	≥5.5 h	≥7.5 h

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 (>10⁸ colony forming units (CFU) per microgram DNA; see figure " Transformation without recovery incubation").

See figures

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

Transformation without recovery incubation.

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 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 (>10⁸ colony forming units (CFU) per microgram DNA; see figure "Transformation without recovery incubation").

Components included with QIAGEN PCR Cloning Kits

Component	QIAGEN PCR Cloning Kit	QIAGEN PCR Cloning^plus Kit	Concentration
pDrive Cloning Vector	+	+	50 ng/µl
Ligation Master Mix	+	+	2x solution
Distilled water	+	+	–
QIAGEN EZ Competent Cells	–	+	–
SOC medium	–	+	–

See figures

pDrive Cloning Vector.

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.

See figures

The QIAGEN PCR Cloning plus Kit procedure.

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.

Comparison of QIAGEN PCR Cloning Kits

Features	QIAGEN PCR Cloning Kit	QIAGEN PCR Cloning^plus Kit
Applications	Cloning of A overhang PCR products	Cloning of A overhang PCR products
Competent cells included	–	QIAGEN EZ Competent Cells
Overhang	U overhang	U overhang
Reaction type	UA hybridization	UA hybridization
Vector included	pDrive Cloning Vector	pDrive Cloning Vector

Supporting data and figures

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

The effect of ligation time on cloning efficiency was compared for the QIAGEN PCR Cloning^plus 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 Cloning^plus Kit procedure set at 100% for each comparison. The ligation was performed for 30 min (QIAGEN PCR Cloning^plus Kit recommendation).

Resources

For QIAGEN PCR Cloning Kit QIAGEN PCR Cloningplus Kit

Addressing critical factors and new solutions

Publications

A novel human B cell subpopulation representing the initial germinal center population to express AID.

Kolar GR; Mehta D; Pelayo R; Capra JD;

Blood; 2006; 109 (6):2545-52 2006 Nov 28 PMID:17132718

Bacterial population dynamics in dairy waste during aerobic and anaerobic treatment and subsequent storage.

McGarvey JA; Miller WG; Zhang R; Ma Y; Mitloehner F;

Appl Environ Microbiol; 2006; 73 (1):193-202 2006 Nov 3 PMID:17085701

Role for nonstructural protein 1 of severe acute respiratory syndrome coronavirus in chemokine dysregulation.

Law AH; Lee DC; Cheung BK; Yim HC; Lau AS;

J Virol; 2006; 81 (1):416-22 2006 Oct 11 PMID:17035307

Characterization of a novel 5' subgenomic RNA3a derived from RNA3 of Brome mosaic bromovirus.

Wierzchoslawski R; Urbanowicz A; Dzianott A; Figlerowicz M; Bujarski JJ;

J Virol; 2006; 80 (24):12357-66 2006 Sep 27 PMID:17005659

Population dynamics within a microbial consortium during growth on diesel fuel in saline environments.

Kleinsteuber S; Riis V; Fetzer I; Harms H; Müller S;

Appl Environ Microbiol; 2006; 72 (5):3531-42 2006 May PMID:16672500

FAQ

The longest fragment we have cloned with this system was about 3 kb. However, some of our customers have successfully cloned 5 kb fragments with the pDrive Cloning Vector of the QIAGEN PCR Cloning Kit. Since the two systems are very similar, we would encourage you to give it a try if you have fragments longer than 3 kb. When using such long inserts, ligation time should be at least 2 hours to increase cloning efficiency.

-30

No. Based on ligation and religation experiments, QIAGEN has not observed any instability of the U-overhang following multiple freeze/thaw cycles. Therefore, it is unnecessary to aliquot the pDrive Cloning Vector to avoid freeze/thaw cycles.

FAQ ID -323

PCR products that will be cloned using the QIAGEN PCR Cloning Kit should be generated using a thermostable DNA Polymerase without proofreading activity, such as Taq DNA Polymerase. Such polymerases attach a single A overhang to their reaction products, which can hybridize to the U overhang of the pDrive Cloning Vector. For efficient addition of an A overhang during the PCR procedure, we recommend a final extension step for 10 min at 72°C as described in the standard protocols of the Taq PCR- and HotStarTaq PCR handbook.

FAQ ID -165

Tables with a complete listing of all restriction sites for the pDrive Cloning Vector of the QIAGEN PCR Cloning Kit are provided in the Appendix 'pDrive Cloning Vector restriction sites' of the QIAGEN PCR Cloning Handbook. The vector is supplied in linear form with a U-overhang at each end. Insertion of a PCR product destroys the EcoRV site of the vector. Therefore, use of EcoRV for restriction mapping of recombinant plasmid is not recommended. Visit our website for the complete pDrive Cloning Vector sequence.

FAQ ID -160

At this time, the Qiagen EZ Competent Cells are not sold separately. They are available only as a component of the QIAGEN PCR Cloning Plus Kit.

FAQ ID -763

The QIAGEN EZ Competent Cells are dam +.

FAQ ID -638

Genotype of QIAGEN EZ Competent Cells supplied in the QIAGEN PCR Cloning plus Kit:

Genotype	Description/phenotype
F'	Presence of the low-copy-number F plasmid [F'::Tn10(Tc^r) proA⁺ B⁺ lacI^q Z delta-M15]
Tn10 (Tc^r)	Transposon conferring tetracyclin resistance
lacI^q	Overproduction of the lac operon repressor, which controls expression from P_lac
lacZ delta-M15	Expression of N-terminally deleted beta-galactosidase.This protein and LacZ alpha-peptide (encoded by the pDrive Cloning Vector) provide alpha-galactosidase activity. Cells transformed by pDrive Cloning Vector which does not contain a PCR product will express LacZ alpha-peptide, and will form blue colonies when grown in the presence of X-gal/IPTG. In contrast, cells transformed by pDrive Cloning Vector which does contain a PCR product will not express LacZ alpha-peptide, and will form white colonies.
recA1	Abolished homologous recombination
end A1	Abolished nonspecific endonuclease I activity, and hence improved DNA quality in plasmid preparations
hsdR17	This mutation prevents restriction, but not protective methylation, of unmethylated DNA or DNA containing foreign methylation patterns in E. coli cells.
lac	Inability to utilize lactose
glnV44	Supressor of amber (UAG)mutation; required for growth of some phage vectors. Formerly called supE
thi-1	Requires thiamin (vitamin B1) for growth in minimal medium
gyrA96	Mutation in DNA gyrase; resistance to naladixic acid
relA1	Relaxed mutation, permits RNA synthesis in the absence of protein synthesis

ta ua pcr cloning

FAQ ID -157

Light blue colonies or blue colonies can sometimes be positive clones. If the LacZ alpha fragment and the cloned insert are in frame, LacZ complementation can occur resulting in blue colonies. Both white and light blue colonies on the same plate can result when the insert is cloned in either orientation and read through only occurs in one orientation. Incubating the plate overnight at 4°C will enhance the blue color facilitating the selection of white or light blue colonies.

FAQ ID -512

The pDrive cloning vector contains the standard sequencing annealing sites for M13 forward and reverse, as well as T7 and SP6 primers. Figure 1 in the QIAGEN PCR Cloning Kit Handbook shows the pDrive Cloning Vector Map. The positions of the T7 and SP6 promoter sites and the M13 forward and reverse sequencing primer binding sites are provided. You can find the primer sequences at the end of the Appendix in this handbook.

FAQ ID -292

Light blue colonies or really blue colonies obtained using the QIAGEN PCR Cloning Kit can also be positive clones. Sometimes, the LacZ alpha fragment and the cloned insert are in frame, resulting in a protein with beta-galactosidase activity. As this protein might not be very active and the X-gal may not be digested as rapidly, colonies may appear only light blue. The likelihood for this to occur increases with larger inserts.

FAQ ID -603

The components of the SOC medium are:

0.5% Yeast Extract
2% Tryptone
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
20 mM Glucose*

*Note: add Glucose after autoclaving the solution with the remaining ingredients, and letting it cool down. Sterilize the final solution by passing it through a 0.2 µm filter.

SOC medium can be stored at room temperature and is stable for several years.

FAQ ID -798

Yes, it is possible to use the pDrive Cloning Vector from the QIAGEN PCR Cloning Kit for in-vitro transcription. It contains SP6 and T7 polymerase promoters. However, we do not have a special protocol for this purpose. We recommend to varify insert orientation via sequencing. Note that the pDrive Cloning Vector does not contain transcription terminators. Therefore, we recommend to linearize the vector at the end of the insert to create a stop point for RNA Polymerase. To overcome the very high template affinity of this enzyme, use a cutter that provides a 5' overhang, thereby promoting that it falls off the template. The T7 transcription start site is located within the promoter region, leading to transcription of the last three guanidine bases of the promoter. When planning to use the in-vitro transcript as a standard for RT-PCR, note that it will also contain truncated products.

FAQ ID -332

QIAGEN offers the pDrive Cloning Vector for direct cloning of PCR products. The vector is supplied prelinearized with a 3'-U overhang. This U overhang hybridizes with high specificity to the 3'-A overhang of PCR products generated by Taq and other non-proofreading DNA polymerases. The pDrive Cloning vector is a component of the QIAGEN PCR Cloning Kit, and has several features designed to facilitate analysis of cloned PCR products.

FAQ ID -146