Geneious Prime provides all of the tools required for primer design and in silico simulation of Gateway® cloning.
ThermoFisher describe their Gateway® Technology as "a universal cloning method that takes advantage of the site-specific recombination properties of bacteriophage lambda to provide a rapid and highly efficient way to move a gene of interest into multiple vector systems." See the Thermofisher website for a full description of Gateway® cloning.
To express a gene of interest using Gateway the following steps are taken:
Generate an entry clone by performing a BP recombination reaction between a pDONR™ vector and an attB PCR product or expression clone.
Generate the desired expression clone by performing an LR recombination reaction between the entry clone and a Gateway® destination vector.
Introduce your expression clone into the host system of choice for expression of your gene of interest.
In this tutorial we will learn how to:
Design Gateway-compatible oligonucleotide primers to amplify a sequence of interest
Simulate PCR to create a Gateway-compatible PCR product
Simulate recombination of PCR product with a donor vector via a "BP" reaction to create an Entry vector
Simulate recombination of an Entry vector with a Destination vector via an "LR" reaction to create an Expression vector
Simulate multipart Gateway cloning
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Click on the Overview link below to start this tutorial.
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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