#Serial cloner build construct how to

If one or more of your sequences contain type IIS restriction sites that you do not want be involved in the assembly then you will need to engineer each site out of your fragment, taking care to avoid altering any gene product sequences.Įxercise: Assembly of 6 Parts into a vector If one or more of your sequences contain the specified type IIS restriction site/s then Geneious will assume you want to use the site/s in the assembly process and design a strategy accordingly. Removal of unwanted internal Type IIS sites

#Serial cloner build construct how to

See Part 2 of this exercise, section Checking the correct primer_bind boundaries are used, for information on how to do this. You have the option to ignore or choose alternate primer_bind annotations associated with each Part. Overriding the default use of primer_bind annotations as Part fusion points (Rules 4 & 5) The fusion point will be the terminus of the blunt end fragment.

If Geneious finds a blunt end, and no suitable type IIS sites or primer_bind annotations are present, then a primer with an appropriate type IIS site extension will be designed.

If a primer_bind annotation without an extension is found, then an extension will be appended to introduce a valid type IIS recognition site, resulting in a new primer sequence.

Existing primer_bind annotation(s) without extension(s)

If Geneious detects a primer annotation with an extension which does not contain a valid type IIS site then the 5′ terminus of the extension will be considered the fusion point and the extension will be extended to introduce a valid type IIS recognition site, resulting in a new primer sequence.ĥ.

Existing primer_bind annotations with extension However, a second “opposite orientation” primer will be designed, based on the appropriate rule, for PCR.Ĥ.

If Geneious detects a single primer_bind annotation with a suitable type IIS site Geneious will assume you wish to use it and a new primer will not be designed.

Geneious will assume that you already have the corresponding primers, and new primers will not be designed for the region.

If Geneious detects a pair of inward facing primer_bind annotations with valid compatible type IIS sites then it will assume you wish to use them.

Existing primer_bind annotation(s) with valid type IIS cut site(s) on the extension Geneious will also assume that you have this “sticky ended” DNA available and so will not design primers for PCR.ģ.

If Geneious detects a pair of valid overhangs compatible with the specified type IIS site, then it will assume you wish to use them.

A second “opposite orientation” primer for PCR will be designed based on rules 3-6. A primer will be designed which incorporates the site.

If only one type IIS cut site is detected, then Geneious will assume you wish to use it.

Geneious will also assume that you have DNA available to use, and so will not design primers for PCR.

If Geneious detects a pair of appropriately orientated type IIS sites with unique overhangs, then it will assume you wish to use them.

If required, Geneious will then design a primer pair for PCR amplification of each Part. Geneious will then choose one, or a combination of these, in order of precedence (see rules 1 to 6 below) to define the insert boundaries to be used for Golden gate recombination. Geneious will analyse your backbone (if defined), and each sequence passed to it, and will detect existing type IIS restriction sites, overhang annotations, primer_bind annotations and blunt ends. Note that the Geneious Golden gate tool currently does not allow the use of multiple type IIS restriction enzymes for assembly. Usually the “receiving” type IIS sites already present in your backbone will define the site you will use. The following rules apply regardless of the type IIS enzyme selected for assembly. Prior to using the tool you should decide which type IIS restriction enzyme you are going to use. Golden Gate in Geneious Prime: The Basics