14.5 Restriction Cloning

Use the Restriction Cloning operation to ligate two or more linear or circular nucleotide sequences. For an overview of the restriction cloning interface, see section 14.4 .

To run a Restriction Cloning operation, optionally select a Backbone sequence, along with one or more Insert sequences, and then specify the enzymes you wish to consider for the reaction. You can adjust the order in which the sequences will be ligated, and the cut sites to be used for the ligation in the Construct Layout Panel.

Candidate Enzymes:

Select Annotated Enzymes if the restriction sites for your reaction are already annotated on your sequences. Enzymes annotated on any of the selected sequences will be available for all sequences in your reaction. To also use restriction enzymes which have not been annotated on your sequences, select Enzyme Set. If None is selected, only blunt end ligation or annotated single-stranded overhangs (‘sticky ends’) will be available. Annotated overhangs and blunt end ligation will remain available if either Annotated Enzymes or Enzyme Set are selected. Restriction enzymes with recognition sites shorter than 5 bp will only be considered if they have been annotated. Contact Geneious Support if you want to change this minimum recognition site length.

Enzyme Set:

This option is enabled when Enzyme Set is selected under the Candidate Enzymes option. All enzyme sets in your database will be available here. You can create new enzyme sets from the Enzymes tab in the Sequence Viewer.

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 change ligation reactions or reverse complement a sequence, click on the ▾ at the right of the sequence tag:

5’ Reaction / 3’ Reaction:

Enzymes from the specified Candidate Enzymes will be shown in the appropriate section, if they have cut sites that are compatible with a neighbor. If more than 30 restriction sites are available, click on Choose enzyme cut site … to select from the available restriction sites. Sites shown in grey italics can be selected, but will require further changes to create a valid reaction. Predigested overhangs and blunt ends are also shown.

Reverse Complement:

Select this option to reverse complement the current sequence, and recalculate possible compatible cut sites on the current sequence and its neighbours. The original sequence will not be altered. Names of reverse complemented sequences appear in blue.

Modify Overhangs:

Select this option to modify overhangs after choosing restriction sites. This allows you to simulate end repair by polymerase-mediated backfilling or nuclease-mediated overhang removal (blunting). Modified overhangs will be retained until you select a new restriction enzyme or use the Reverse Complement option.