PlasmidLab / Guides / Gateway® cloning

Gateway® cloning: what BP and LR do

Gateway® moves a segment between plasmids by recombination, with nothing cut and nothing ligated. Two reaction names cover the whole system.

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Ask the agent

Open the source, the donor and the destination vector, and ask for the route through both reactions.

Get the GFP from the pUC19 clone into pPL-DEST, by way of an entry clone in pPL-ENTR. Tell me what to plate on at each step.

The PlasmidLab window with pUC19-GFPmut3b.gb, pPL-ENTR.gb and pPL-DEST.gb open and a request to get the GFP into pPL-DEST by way of an entry clone, with what to plate on at each step.
Three files open, and the request.

The agent designed the attB-tailed primers, ran the BP reaction into the donor to make the entry clone, then the LR into the destination, and wrote both plasmids out as tabs. It also answered the part of the question the menus cannot: what to plate on after each step, and the warning that a destination vector grown in a ccdB-resistant strain gives a lawn of empty vector.

The reply in the AI panel next to the product tab pPL-DEST-GFPmut3b, 3,197 bp, with the reply confirming the GFP translation is identical to the source and the product is circular.
Both clones, and what to plate on.
The agent works through PlasmidLab's own cloning tools, so what it makes is an ordinary file you can open, edit and save. Your sequences go to the AI provider you pick; the privacy page says what is sent.

Or do it yourself

Clone > Gateway® Cloning… runs BP, LR, or both in one pass, and writes out the entry clone it made on the way as well as the expression clone.

The Clone menu open over the destination vector map, with Gateway Cloning listed among the cloning methods.
Gateway sits with the other methods.

What the two reactions are

BP joins attB and attP and leaves attL and attR: an insert with attB ends plus a donor vector gives an entry clone. LR joins attL and attR and leaves attB and attP: the entry clone plus a destination vector gives the expression clone. One entry clone can go into as many destination vectors as you like, which is the point of the system.

Sites come in numbered pairs, 1 with 1 and 2 with 2, so the insert can only land one way round. Donor and destination vectors carry ccdB between their sites; its product poisons DNA gyrase, so only the recombined plasmid forms colonies, and the vectors themselves have to be kept in a ccdB-resistant strain. An att site ends up between a vector tag and your insert and is translated, so for an N-terminal tag the frame has to run through attB1 into your coding sequence.

Before you order

Read the product before you spend money on oligos: the sequence at each junction, the reading frame through a coding fusion, and the feature orientation. A design is not experimental validation, and the agent can be wrong.