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Cloning & Competent Cells

Products >  Cloning_and_Competent_Cells >  Cloning_Resources >  Primer_Design_Tutorial >  Primer_Design_Overview

Designing Primers for In-Fusion Cloning

The Online In-Fusion Primer Design Tool allows you to generate:

  • Insert-specific PCR primer sequences for In-Fusion cloning
  • A diagram of your In-Fusion cloning experiment, including your vector and insert sequences, and displaying either the primer sequences generated by the tool or primer sequences of your own design
  • A text file containing the entire sequence of your recombinant construct, which can be entered into any nucleotide sequence analysis software tool

You can use this tool with six different types of vector/insert combinations, as described below.

Select Your Protocol:

  • General Considerations Before Beginning In-Fusion Primer Design
    1. Terminology: The following terms are used interchangeably throughout this protocol:
      • “insert,” “fragment,” “insert fragment,” and “cloning fragment”
      • “In-Fusion primer,” “In-Fusion PCR primer,” and “fragment-specific PCR primer”
    2. Browser compatibility: The Online In-Fusion Primer Design Tool is compatible with the Mozilla Firefox and Google Chrome browsers, but not with Internet Explorer.
    3. Entering sequence information: The nucleotide sequences of the vector and insert should be entered in plain text format without nucleotide or line numbering.

      IMPORTANT! If you must go back to a previous page after entering data, be sure to use the Primer Tool’s “Go Back” button located near the bottom center of your current page, instead of the “Back” arrow in your browser, or your data will be lost.

    4. Minimum insert length: 50 bases
    5. Cloning an insert in-frame with a tag: If you are inserting a cloning fragment in the same translational reading frame as a tag (e.g. fluorescent protein, Myc, or HA), we recommend adjusting the sequence of the corresponding insert-specific PCR primer to ensure reading frame continuity. Be careful not to introduce intervening stop codons when you design your primers.
  • Tips for PCR Amplification, Using In-Fusion PCR Primers:
    1. To ensure PCR amplification:
      The insert-specific portion of each In-Fusion PCR primer should be 18–25 bases long.
    2. To optimize PCR amplification:
      Perform the initial 3–5 PCR cycles using the optimum annealing temperature for the insert-specific 3'-end of the In-Fusion PCR primer.
      • The annealing temperature for the remaining PCR cycles should be optimized as a balance between the insert-specific 3'-end of the primer and the entire primer.
      • The melting temperature of the entire primer may be rather high—so you should optimize the annealing temperature to allow efficient PCR amplification.
    3. In case of inefficient PCR amplification:
      In-Fusion PCR primers may require redesigning by extending or, if necessary, repositioning the insert-specific 3'-end of the primer.

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