+1 877 302 8632
+1 888 205 9894 (Toll-free)

PCR Cloning Kit (CloneEZ)

Pubmed (34 references)
Catalog No. ABIN491483
Plus shipping costs $45.00
24 reactions
Shipping to: United States
Delivery in 4 to 6 Business Days
  • Application
    Cloning (Clon)
    CloneEZ® PCR Cloning Kit is designed for quick and convenient PCR cloning.
    CloneEZ® PCR Cloning Kit is designed for the quick PCR cloning , it is especially powerful in high-throughput cloning of PCR products into any destination vector effectively by sidestepping the tedious and limiting tasks such as selecting proper restriction enzymes, phosphatases, or ligases. Using our proprietary CloneEZ® Enzyme, this kit is able to rapidly generate precise and directional constructs by carrying out 30-minute incubation at room temperature (20 °C-25 °C).
    CloneEZ® Enzyme (5 U/µL): 50 µL
    10X CloneEZ® Buffer: 100 µL
    pUC57 Linearized with EcoRI/HindIII, positive control: 10 µL
    1-kb Control Insert, positive control: 10 µL
  • Comment

    The CloneEZ® PCR Cloning Kit offers flexible and quick directional cloning and enables users to:
    Clone into any vector without any need for restriction enzymes, phosphatase treatments, or ligases
    Provide up to 10 kb PCR cloning at any restriction site
    Rapidly and precisely generate correctly oriented constructs and inserts
    Work with PCR that employ any thermostable polymerases

    Assay Time
    < 1 h
    Reagent Preparation

    To clone any DNA fragment into a linearized vector using this kit, the insert fragment should be obtained by PCR using primers with an add-on of 15 base sequences homologous to either side of the restriction site that is used to linearize the vector. Therefore, a primer should cover a 15-base sequence add-on at the 5’-end, an optional restriction site in the middle, and the insert-specific sequence at the 3’-end. PCR amplification can be performed using any thermostable DNA polymerase. However, primers and primer dimers produced in PCR reactions are inhibitory to the CloneEZ® PCR cloning reaction. If the PCR produces a single specific band (from an agarose gel), PCR DNA can be purified by simply using a PCR purification kit.

    Sample Preparation

    To achieve a successful CloneEZ® PCR cloning reaction, complete linearization of the vector is critical. Incomplete linearization of the vector will result in high background. The linearized vector should be purified using a gel or PCR purification kit, such as QuickClean I PCR Purification Kit

    Assay Procedure

    CloneEZ® Recombination Procedure
    1. Set up the following reaction in a 0.5 mL Eppendorf tube by mixing the following reagents gently and then spin down briefly to collect the reagents at the bottom of the tube.
    Linearized vector (100-200 ng/µL): 6 µL
    Purified PCR products (100-200 ng/µL): n µL
    10X CloneEZ® Buffer: 2 µL
    CloneEZ® Enzyme: 2 µL
    Deionized water: up to 20 µL In general, add more than 10 µL of PCR DNA (n = 10) to the reaction can produce nearly 95% positive clones. In addition, less amount of DNA is appropriate for short PCR DNA fragments. For different sizes of PCR DNA, different amount of DNA is recommended below: PCR DNA of 1 kb: 4 µL
    PCR DNA of 2 kb: 6 µL
    PCR DNA of 3 kb: 8 µL
    PCR DNA of >3 kb: 10 µL
    2. Incubate the reactions at 22°C for 30 minutes, and then transfer tubes to ice and incubate on ice for five minutes.
    3. Proceed with transformation (Section D). The reaction can also be stored at -20°C for later transformation.

    Materials needed but not provided along with the kit:
    Water bath or heating block (42°C)
    SOC liquid mediumDH5α competent cells (>1×108 cfu/µg)
    1. Thaw one vial of frozen 50 µL competent cells on ice. Tap the tube gently to ensure that the cells are suspended.
    2. Add 5–8 µL of reaction mixture to the competent cells. Tap the tube gently and incubate the tube on ice for 30 minutes.
    3. Heat shock the cells by placing them in 42oC water bath for 45–90 seconds and then place the tube on ice for 2–3 minutes.
    4. Add 600 µL of SOC medium to the cells and then incubate the cells on a shaker set at 250 rpm at 37 °C for 60 minutes.
    5. Centrifuge the cell down at 4000 rpm for five minutes and then remove and discard about 500 µL of medium. Gently suspend the cells by tapping the tube.
    6. Transfer 10 µL and 100 µL of the suspension to two different plates containing appropriate antibiotics, respectively. Spread the cells evenly on the plates.
    7. Incubate the plates overnight at 37 °C.

    For Research Use only
  • Storage
    -20 °C
    Storage Comment
    The kit should be stored at -20°C. It will remain stable for at least one year.
    Expiry Date
    12 months
  • Wei, Oh, Million, Cate, Jin: "Simultaneous utilization of cellobiose, xylose, and acetic acid from lignocellulosic biomass for biofuel production by an engineered yeast platform." in: ACS synthetic biology, Vol. 4, Issue 6, pp. 707-13, (2015) (PubMed).

    Wang, Zhu, Chen, Wang: "mRNA m?A methylation downregulates adipogenesis in porcine adipocytes." in: Biochemical and biophysical research communications, Vol. 459, Issue 2, pp. 201-7, (2015) (PubMed).

    Zeng, Liu, Wang, Fang: "A bispecific antibody directly induces lymphoma cell death by simultaneously targeting CD20 and HLA-DR." in: Journal of cancer research and clinical oncology, Vol. 141, Issue 11, pp. 1899-907, (2015) (PubMed).

    Tang, Zhou, Zhang, Xiao, Hu, Zhang, Huang, Chen, Liu, Liang: "Synergetic action of domain II and IV underlies persistent current generation in Nav1.3 as revealed by a tarantula toxin." in: Scientific reports, Vol. 5, pp. 9241, (2015) (PubMed).

    Liu, Cui, Liu, Cui, Xia, Kobayashi, Zhou: "Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide." in: Journal of bioscience and bioengineering, Vol. 118, Issue 3, pp. 249-52, (2014) (PubMed).

    Hui, Al Zaki, Cheng, Popik, Zhang, Luning Prak, Tsourkas: "Facile method for the site-specific, covalent attachment of full-length IgG onto nanoparticles." in: Small (Weinheim an der Bergstrasse, Germany), Vol. 10, Issue 16, pp. 3354-63, (2014) (PubMed).

    Wang, Tan, Jiao, You, Zhang: "Analyzing cold tolerance mechanism in transgenic zebrafish (Danio rerio)." in: PLoS ONE, Vol. 9, Issue 7, pp. e102492, (2014) (PubMed).

    Elias, Crayton, Warden-Rothman, Tsourkas: "Quantitative comparison of tumor delivery for multiple targeted nanoparticles simultaneously by multiplex ICP-MS." in: Scientific reports, Vol. 4, pp. 5840, (2014) (PubMed).

    Wang, Yomano, Lee, York, Zheng, Mullinnix, Shanmugam, Ingram: "Engineering furfural tolerance in Escherichia coli improves the fermentation of lignocellulosic sugars into renewable chemicals." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, Issue 10, pp. 4021-6, (2013) (PubMed).

    Kato, Aoyama, Maeshima: "The Ca(2+) -binding protein PCaP2 located on the plasma membrane is involved in root hair development as a possible signal transducer." in: The Plant journal : for cell and molecular biology, Vol. 74, Issue 4, pp. 690-700, (2013) (PubMed).

    Wang, Galan, Normandin, Bonneil, Hickson, Roux, Thibault, Archambault: "Cell cycle regulation of Greatwall kinase nuclear localization facilitates mitotic progression." in: The Journal of cell biology, Vol. 202, Issue 2, pp. 277-93, (2013) (PubMed).

    Dondelinger, Aguileta, Goossens, Dubuisson, Grootjans, Dejardin, Vandenabeele, Bertrand: "RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition." in: Cell death and differentiation, Vol. 20, Issue 10, pp. 1381-92, (2013) (PubMed).

    Chen, Xu, Shen, Yan, Xu, He, Ma: "Engineering arsenic tolerance and hyperaccumulation in plants for phytoremediation by a PvACR3 transgenic approach." in: Environmental science & technology, Vol. 47, Issue 16, pp. 9355-62, (2013) (PubMed).

    Nie, Liu, Zhang, Zhao, Fan, Ning, Zhang: "Production and secretion of Lactobacillus crispatus ?-galactosidase in Pichia pastoris." in: Protein expression and purification, Vol. 92, Issue 1, pp. 88-93, (2013) (PubMed).

    Pardini, Rosa, Barone, Di Gaetano, Slyskova, Novotny, Levy, Garritano, Vodickova, Buchler, Gemignani, Landi, Vodicka, Naccarati: "Variation within 3'-UTRs of base excision repair genes and response to therapy in colorectal cancer patients: A potential modulation of microRNAs binding." in: Clinical cancer research : an official journal of the American Association for Cancer Research, Vol. 19, Issue 21, pp. 6044-56, (2013) (PubMed).

    Mao, Cheng, Lei, Xu, Gao, Ren, Wang, Zhang, Wang, Wu, Guo, Liu, Wu, Wang, Wan: "Wax crystal-sparse leaf2, a rice homologue of WAX2/GL1, is involved in synthesis of leaf cuticular wax." in: Planta, Vol. 235, Issue 1, pp. 39-52, (2012) (PubMed).

    Liu, Wang, Wang, Feng, Zhu, Wang: "Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility." in: PLoS ONE, Vol. 7, Issue 1, pp. e29875, (2012) (PubMed).

    Wang, Ma, Zhu, Li, Du, Chen: "Available methods for assembling expression cassettes for synthetic biology." in: Applied microbiology and biotechnology, Vol. 93, Issue 5, pp. 1853-63, (2012) (PubMed).

    Tang, Fares, Zhao, Du, Liu: "Different endocytic functions of AGEF-1 in C. elegans coelomocytes." in: Biochimica et biophysica acta, Vol. 1820, Issue 7, pp. 829-40, (2012) (PubMed).

    Spiliotis: "Inverse fusion PCR cloning." in: PLoS ONE, Vol. 7, Issue 4, pp. e35407, (2012) (PubMed).

You are here: