Primer Design Tutorial

1. Enter the Vector Sequence and Select Restriction Site(s) for Linearization
   1. Select the vector configuration (type):
      • Circular > Linearized by Restriction Enzyme (circled in red)
        
   2. Enter the nucleotide sequence of the cloning vector in the “Vector” box, in plain text format without nucleotide or line numbering.
      
   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 the primers that preserve these restriction sites in your final construct, click the “Preserve restriction site(s)” checkbox (marked by the red “X”).

      
      

      NOTES:

      If you want to de-select an undesired restriction enzyme, highlight its name in the small box to the right of the “Select Restriction Enzyme” box, and then click on the reverse arrow “<<”. Note that this enzyme will then appear to have disappeared from the “Select Restriction Enzyme” box, but will in fact have moved to the bottom of the list (you will need to scroll down to find it—see example below).

      
   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.
      
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).
      
   2. Enter your first insert sequence into the “Insert Fragment” box. The minimum sequence length is 50 bases.
      
      To add an additional insert sequence, click the “Add Fragment Item (Option)” button, indicated by the red arrow and enter your next insert sequence into the new “Insert Fragment” box that appears underneath the first box.
      
   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 all 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.

          IMPORTANT!

          • If you choose to indicate the nucleotide positions of the primers instead of entering the primer sequences, you must do this for all of the fragments you enter.
          • Make sure not to include any in-frame stop codons in the 3' primer for your first cloning fragment.

        
        
      • 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.

        
        
   4. Once you have entered the sequences of all the cloning fragments and fragment-specific PCR primers (or omitted these to let the tool design your 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.
      
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
      
   2. In-Fusion cloning diagram
      
   3. Homologous overlaps between the cloning fragments and the vector
      
   4. Homologous overlaps between each pair of adjacent cloning fragments
      
   5. 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.

      
      

      NOTE:

      • Click the “All Sequence of Cloning construct” button to obtain a text file of the sequence of your In-Fusion construct.
      • Click the “Cloning Diagram” button to display the diagram alone (see example below).

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