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Restriction Cloning

The Geneious Prime Restriction Cloning tool can ligate any combination of linear or circular nucleotide sequences, will automatically identify compatible cut sites, and perform one-step digestion and ligation with drag and drop ordering of two or more fragments.

Input documents do not need to be predigested or annotated with restriction sites, as Geneious will identify the restriction sites present in your input documents and attempt to guess the sites your cloning strategy will use.  By default, restriction sites with recognition sites of 4 bp or less are not considered.  If you need the Restriction Cloning tool to use 4 bp cutters then contact the Geneious support team.

The Restriction Cloning tool can be found under menu Tools → Cloning, or via the Cloning button on the Toolbar. You can launch the Restriction Cloning tool with or without selecting your input sequences first.

In this tutorial we will ligate a PCR product into the multiple cloning site (MCS) of the expression vector pET26B (See Novagen). The pET26B vector MCS lies within a short coding sequence (CDS) that encodes an N-terminal PelB signal sequence for potential periplasmic localization and a C-terminal His•Tag® sequence.


The PCR product provided in this tutorial has been generated with PCR primers designed to amplify the region encoding the mature XynA enzyme (without signal peptide or stop codon) from the bacterium Dictyoglomus thermophilum.  The primers were designed with extensions to generate an N-terminal NcoI site and a C-terminal BamHI site.  The NcoI site has been positioned so that ligation will give an in-frame fusion to the pET26B-based PelB signal peptide.  The BamHI site is positioned so that ligation will generate an in-frame C-terminal fusion of XynA to the pET26B-based HIS-tag.

Go to Part 1 to start the tutorial.

Part 1: Creating restriction enzyme subsets

Part 2: Performing Restriction Cloning

Part 3: Confirming CDS in-frame fusion

Part 4: Troubleshooting