The first step in Gateway cloning is the preparation of a Gateway Entry clone. There are a few different ways to make entry clone. 
# Gateway attB1 and attB2 sequences are added to the 5 and 3 end of a gene fragment, respectively, using gene-specific PCR primers and PCR amplification. The PCR amplification products are then mixed with a proprietary mixture of plasmids called Gateway "Donor vectors" (Invitrogen terminology) and proprietary "BP Clonase" enzymes. The enzyme mix catalyzes the recombination and insertion of the PCR product containing the attB sequence into the attP recombination sites in the Gateway Donor vector. When the cassette is part of the target plasmid, it is referred to as an "Entry clone" in Gateway nomenclature and the recombination sequences are referred to as Gateway "attL" type.
# A short end containing attL is added using the TOPO method, a technique in which DNA fragments are cloned into specific vectors without the need for DNA ligases.
# The desired DNA sequence can be cloned into a multicloning site containing attL using restriction enzyme.
The second step in Gateway cloning is the preparation of a Gateway Destination vector. It is important to choose the target vector that best suits your target when preparing the expression clone. The gene cassette in the Gateway Entry clone can then be simply and efficiently transferred into any Gateway Destination vector (Invitrogen nomenclature for any Gateway plasmid that contains Gateway “attR” recombination sequences and elements such as promoters and epitope tags, but not ORFs) using the proprietary enzyme mix, “LR Clonase”. Thousands of Gateway Destination plasmids have been made and are freely shared amongst researchers across the world. Gateway Destination vectors are similar to classical expression vectors containing multiple cloning sites, before the insertion of a gene of interest, using restriction enzyme digestion and ligation. Gateway Destination vectors are commercially available from Invitrogen, EMD (Novagen) and Covalys.
The third step in Gateway cloning is the preparation of express your gene of interest. Make sure to use sequencing or a restriction digest to check the integrity of your expression clone. Once your construct is working, you can transform or transfect the cells you intend to employ in your investigations.
Since Gateway cloning uses patented recombination sequences, and proprietary enzyme mixes available only from Invitrogen, the technology does not allow researchers to switch vendors and contributes to the lock-in effect of all such patented procedures.
To summarize the different steps involved in Gateway cloning:
* Gateway BP reaction: PCR-product with flanking att B sites (this step can also use other methods of DNA isolation, such as restriction-digestion) + Donor vector containing attP sites + BP clonase => Gateway Entry clone, containing att L sites, flanking gene of interest
* Gateway LR reaction: Entry clone containing att L sites + Destination vector containing att R sites, and promoters and tags + LR clonase => Expression clone containing attB sites, flanking gene of interest, ready for gene expression.