In this tutorial, you will prepare for restriction cloning by creating primer pairs for a gene of interest, and then manually introducing restriction sites.

  1. If you have not yet download and extracted the tutorial data, click here to download it. Then decompress (unzip) the file archive using the method of your choice.
  1. Use File > Open to open the file N.thermophilus_his.sbd. The sequence opens in the Circular view.
  1. Click on the feature named PAS/PAC sensor signal transduction to select it. Notice in the header that the selected range 10086..11369.

  1. Select Priming > Create Primer Pairs. The Create Primer Pairs dialog appears.
  1. Select the radio button next to Choose optimal primer location.

The default settings for this option allow SeqBuilder Pro to choose the best primer locations from within 50 bp of the surrounding sequence.

  1. Click OK. SeqBuilder Pro locates the best primer pair and displays the forward primer in the Primer Design view. The header shows that the forward primer is initially 24 bp in length.

  1. Extend the primer to 32 bp in length by dragging the triangle on the 5’ (left) end further to the left.
  1. Select the Introduce Restriction Site tool ( ) from the Primer Design toolbar to enter into the mode for introducing restriction sites. Notice that the cursor changes to look like scissors.
  1. Place your cursor to the immediate left (5’ end) of the extended primer, between the arrow and the letter ‘C,’ and click.

  1. From the resulting menu, select Any Enzyme, and then type the letter “S” to scroll to enzymes starting with that letter. Click the triangle at the bottom of the list to reveal additional options and then select the enzyme SacII. The SacII enzyme is added to the document.

New restriction sites are displayed in magenta. The same color is used for nucleotides in the primer sequence and their translations that differ from the template. Restriction enzymes whose recognition sites are eliminated by the change are shown in blue.

  1. Click the Show Reverse Primer tool ( ) to display the reverse primer. Notice that the reverse primer is also 24 bp in length, as shown in the Primer Design toolbar.
  1. Extend the reverse primer 6 bp to increase its length to 30 bp. Do this by dragging its right-hand triangle further to the right.

  1. Select the Introduce Restriction Site tool ( ) again, and click between the right-hand triangle and the ‘T’ to its left.
  1. Select Any Enzyme and then AscI. The AscI enzyme is added to the document.
  1. Select Priming > Create Insert by PCR. A new cloning project is created and the PCR amplified insert appears in the Inserts folder.

The menu command extracted the sequence of the PCR product and simulated its amplification with Taq polymerase including the addition of 3’ A overhangs to each end. In this example, you will use restriction cloning, which cleaves the sequence at the designated restriction sites. Thus, the 3’ A overhangs will not affect the resulting clone.

This workflow continues in Try it! – Create and use a PCR insert in restriction cloning.

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