PCR Taq

PCR Cloning Kit

  • What are the differences between the GenBuilder™ kit and the PCR Cloning Kit?

    GenBuilder™ seamless cloning technology offers high efficiency, quick and direct cloning, bypassing all the tedious procedures of traditional cloning that often involve restriction, ligation, and sometimes phosphorylation. This technology works with any DNA sequence and any vector. The only requirement is that the PCR primers have a sequence containing 15 or more nucleotides homologous to the vector sequence.

  • Technical information is not available in the manual, why is that?

    Currently, we are waiting for a patent on GenBuilder. Once the patent is valid a more detailed explanation in the manual will be provided.

  • Why don't I generate sufficient colonies when the GenBuilder™ reaction is transformed?

    This may be caused by four factors.

    1. The competent cells have low transformation efficiency.
    2. Too much reaction mixture is used.
    3. Presence of Inhibitory contaminants from PCR DNA or linearized vector.
    4. The molar ratio of vector to insert is off.
      Please refer to the manual for TROUBLESHOOTING.
  • Why do I generate incorrect colonies when the GenBuilder™ reaction is transformed?

    Two factors may be involved.

    1. The cloning vector is not completely linearized.
    2. The cloning reaction is contaminated with plasmids having the same antibiotic resistance.
  • What factors need to be considered when designing the primer?

    Two sets of primers are used to amplify the gene of interest:

    1. 15-bp homology regions to the vector, flanking the cloning site into the insert.
    2. Specific gene sequence. If the sequence has 5' overhang, the start of the 15 bp homology must begin from the 5' most extension to include the overhang; if the sequence has a 3' overhang, homology should begin where the DNA becomes double-stranded. Please refer to the manual for illustration of primer design.
  • What should the purity of my primer be to be compatible with the GenBuilder™ cloning method?

    Desalted oligos (from a qualified supplier) are suitable for cloning with the GenBuilder™ PCR cloning Kit.

  • Can multiple fragments be cloned into a single vector using this kit?

    Yes. GenBuilder™ PCR Cloning Kit can be used for multiple fragment recombination if there are not many repeated sequences among multiple fragments. However, we recommend recombination one fragment at a time. Several performances of recombining 2 - 3 fragments demonstrated efficiency as low as 20%-30%, and lead to a direct repeat deletion.

  • Can I use GenBuilder™ PCR Cloning Kit with my own vectors?

    Yes. GenScript's GenBuilder™ PCR Cloning Kit has been successfuly tested with several commercial and non-commercial vectors.

  • What factors need to be considered when using GenBuilder™ PCR Cloning Kit with customers' vector?

    We recommend linearizing your vectors before using GenBuilder™ PCR Cloning Kit. Once linearized, the columned and gel-purified vector is ready for GenBuilder™ reaction.

  • Will the GenBuilder™ reaction work more efficiently if I use primers that contain a longer than 15 base region of homology?

    A 15-20 bp homology is recommended.

  • What is the stability of the enzyme included in GenBuilder™ PCR Cloning Kit?

    The liquid enzyme should be stored at -20°C for at least 12 months without activity loss.

    Problem
    Probable Cause
    Solution
    Few or no colonies are obtained from the transformation.
    The competent cells have low transformation efficiency.
    Check the transformation efficiency. Competent cells with >1×108 cfu/μg are recommended.
    Too much reaction mixture is used.
    Do not add more than 20 μl of reaction mixture to 50 μl of competent cells. Too much reaction mixture inhibits the transformation.
    There are inhibitory contaminants from PCR DNA or from linearized vector. The molar ratio of vector to insert is off.
    Both the PCR DNA and the linearized vector should be purified. Usually an insert/vector molar ratio of 2:1 is optimal. If the insert is as large as the linearized vector, a molar ratio of 1:1 can also be used.
    Too long or short a recombination time.
    It is recommended to keep the recombination procedure within 30 min.
    The linearized cloning vector or primer is large.
    Connect the product to the target step recombinant vector.
    The cloning vector is not completely linearized.
    Gel-purify the linearized vector.
    The cloning reaction is contaminated with plasmids having the same antibiotic resistance.
    Purified PCR DNA may contain the template plasmid, so gel-purify the PCR DNA.
    Most of the colonies contain no insert.
    The cloning vector is not completely linearized.
    Gel-purify the linearized vector.
    The cloning reaction is contaminated with plasmids having the same antibiotic resistance.
    Purified PCR DNA may contain the template plasmid, so gel-purify the PCR DNA.

Plasmid Purification

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