The first three exercises in this tutorial cover the steps required for simulation of single insert Gateway cloning.
The fourth and fifth exercises cover simulation of multisite Gateway cloning.
The final section describes how to prepare vector sequences for use with the Gateway cloning tool.
Thermofisher recommend that to enable efficient Gateway® cloning, primers for addition of attB sites MUST contain the following structure:
When the Geneious Primer design tool is used to add att extensions to primers it will automatically do the following:
† In Geneious Prime 2019.1 and earlier "spacer" nucleotides are added as part of the att motif annotation. In future versions of Geneious Prime the "spacer" nucleotides will be added as separate annotations.
The primer design tool will not add translation initiation motif sequences. Users will need to add these motifs manually as extensions during the primer design process.
The primer design tool will not add a stop codon to a reverse primer. If you require a stop codon you should incorporate a template-derived stop codon (if present) or manually add a stop codon as an extension during the primer design process.
Click the Exercise 1 link below to start the tutorial exercises.
General Overview: Overview of tutorial
Exercise 1: Designing primers for Gateway cloning
Exercise 2: Simulating an "BP" Entry clone reaction
Exercise 3: Simulating a "LR" Destination clone reaction
Multisite Overview: Overview of Gateway multisite cloning
Exercise 4: Multisite Part 1: Primer design and PCR
Exercise 5: Multisite Part 2: Creation of Entry and Destination vectors
Gateway Vectors: Preparing Gateway vector sequences
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