14.6 Golden Gate

Golden Gate is a method to conveniently digest and ligate multiple sequences with the same Type IIS enzyme in a single reaction. For an overview of the cloning interface, see section 14.4 .

To run a Golden Gate cloning operation, optionally select a Backbone sequence, along with one or more Insert sequences, and then specify the enzyme you wish to use for the reaction. You can adjust the order in which the sequences will be ligated and optionally select primers for use in the ligation reaction in the Construct Layout Panel. See Figure 14.6 for an example.

Enzyme:

Select a single Type IIS restriction enzyme to use in the ligation reaction. The set of available enzymes includes those commercially available Type IIS enzymes with an effective recognition site of at least 4 bases which produce an overhang of at least 3 bases on either strand. Only enzymes with 2 or fewer cut sites in each of the selected sequence may be used.

Construct Layout:

Sequences will be displayed as Tags in the Construct Layout Panel, in the order in which they will be ligated. Overhangs for each sequence, corresponding to the selected restriction enzymes, will be displayed between sequence tags. Pairs of overhangs will be green if they are compatible or red if they are not. Unused overhangs that will be generated at either end of a linear construct will be shown in grey.

To select primers or reverse complement a sequence, click on the ▾ at the right of the sequence tag.

Ligation reactions will be shown on the sequence tags as follows:

Pre-digested

if the sequence has existing overhangs on both ends

Cut by [ enzyme ]

if the sequence will be cut with the selected enzyme

PCR Product

if restriction sites for the chosen enzyme will be introduced via PCR and at least one of the existing primers on the sequence is used

Full-length PCR

if newly generated primers will amplify the full sequence and append restriction sites at both ends

Where different reaction types are used at either end ( Overhang, Enzyme or Primer), this will also be indicated