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Cloning & Competent Cells

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Primer Design Tutorial

Cloning a Single Fragment into a Restriction Digest-Linearized Vector

  1. Enter the Vector Sequence & Select Restriction Site(s) for Linearization
    1. Select the vector configuration (type):
      • Circular > Linearized by Restriction Enzyme (circled in red) View example
    2. Enter the nucleotide sequence of the cloning vector in the “Vector” box, in plain text format without nucleotide or line numbering. View example
    3. Select your restriction sites from the “Select Restriction Enzyme” box by highlighting each desired restriction enzyme, and then clicking on the forward arrow “>>” to the right of the box. The selected enzyme names will appear in the smaller box to the right of the arrow.

      IMPORTANT! If you wish to generate primers that preserve these restriction sites in your final construct, click the “Preserve restriction site(s)” checkbox (marked by the red “X”).

      View example
    4. Click the “Get Fragment” button to proceed to the next page of the In-Fusion Primer Design Tool, where you will see the results of your restriction digest and enter the sequence of your cloning fragment. You will also enter the sequences of your fragment-specific PCR primers on this page, or allow the Online In-Fusion Primer Design Tool to design them for you. View example
  2. Enter the Sequences of Your Cloning Fragment and Fragment-Specific PCR Primers
    1. Choose the fragment corresponding to your linearized vector that you will use for cloning (circled in red). View example
    2. Enter your insert sequence into the “Insert Fragment” box. The minimum sequence length is 50 bases. View example
    3. PCR Primers: You can design the primers yourself, or allow the Primer Design Tool to design them.
      • If you are designing the primers yourself, enter the nucleotide sequences of the forward (5') and reverse (3') fragment-specific PCR primers.
        • Enter both primer sequences in the 5' to 3' orientation. The reverse primer sequence should be located in the antisense strand of the cloning fragment.
        • Instead of entering the primer sequences, you may indicate the nucleotide positions of the forward (5') and reverse (3') fragment-specific PCR primers at the desired cloning sites, in the “Position” boxes next to the “Primer” boxes.
      • View example
      • If you are allowing the Tool to design your primers, leave the “Primer” and “Position” boxes blank. The In-Fusion Primer Design Tool will generate these primers for you, incorporating the entire sequence of the entered cloning fragment.

        IMPORTANT! The In-Fusion Primer Design Tool does not allow a combination of user-designed and software-generated fragment-specific PCR primers, so you must enter either all of the primer sequences, or none of them.

        View example
    4. Once you have entered the sequences of your cloning fragment and the fragment-specific PCR primers (or allowed the tool to design primers for you), click the “Go Infusion Primer Design” button to generate a diagram of your In-Fusion PCR primer sequences and your In-Fusion cloning experiment. View example
  3. Information and Primer Sequences Generated by the In-Fusion Primer Design Tool

    NOTE: Overlaps are highlighted in yellow.

    1. In-Fusion PCR primer sequences View example
    2. In-Fusion cloning diagram View example
    3. Homologous overlaps between the cloning fragment and the vector View example
    4. Output: Text files for sequence analysis

      The In-Fusion Primer Design Tool will generate text files of the In-Fusion cloning diagram and the entire nucleotide sequence of the recombinant construct.

      This data can be entered into any nucleotide sequence analysis software tool, such as SnapGene Viewer (available for free online)—which allows easy visualization of In-Fusion primer locations in both the sense and antisense strands.

      IMPORTANT! The melting temperatures of the In-Fusion PCR primers displayed in the In-Fusion cloning diagram are inaccurately high, due to a glitch in the software. For the correct melting temperatures:

      • Refer to the accompanying specification sheet if you order your primers from a vendor.
      • Use an independent software tool for PCR primer analysis, such as OligoAnalyzer 3.1 from IDT Technologies (available for free online).

      NOTE: If you use this tool, enter the standard Mg2+, Na/K, and dNTP concentrations that are usually recommended for PCR, since the reaction buffer composition of CloneAmp HiFi PCR Premix is proprietary.

      View example

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