Q. Can the Geneious Golden gate tool consider multiple type IIS restriction enzymes?
A. No. The Geneious Golden gate tool can only use a single type IIS enzyme.
Q. Why aren't all of the primers designed by the Golden Gate tool annotated onto my final Golden Gate construct?
A. The Golden Gate tool will only map the primer_bind annotations to the output, if the primer_bind annotation matches the sequence 100%. In some cases, for instance when a preexisting type IIS site is utilised, the primer bind region will lie external to the fragment that is assembled, and so will be removed by digestion, and not be present in the final assembly.
Q. Does the Geneious Golden Gate tool exclude adding palindromic overhangs?
A. The Golden Gate tool currently does not exclude using palindromic overhangs.
Q. Does the Geneious Golden Gate tool exclude using overhang combinations where the same three or more consecutive nucleotides are present in another overhang used in the assembly?
A. The Golden Gate tool currently does not check for similarity between overhangs.
Q. Will the Autoarrange button sort based on sequence names?
A. No. The Autoarrange option will try to identify a unique sorting of the sequences based on the available overhangs generated via preexisting sites in your Parts. It does not sort based on sequence name.
Q. I want to have my part synthesized for Golden Gate rather than do PCR - what should I do?
A. Check the option to Save Intermediate products. This will create a PCR product sequence for each Part. Submit this PCR product sequence to you custom synthesis provider of choice. The example below shows the GFP5 PCR product Part that would be generated during the exercise provided in this tutorial.
Q. What criteria are used to design the primer_bind regions of the primers output by the Golden Gate tool? Will they all have similar melting temperatures in the initial rounds of PCR?
A. The Geneious Golden Gate tool uses fairly relaxed settings when designing Golden Gate primers to ensure a primer is designed. The settings used are shown below.
If you wish to design your Golden Gate primers with your own specific primer bind parameters, then you should design and add your own flanking primer_bind annotations to your sequences prior to performing assembly. This will force Geneious to use the annotations as Part boundaries.