How do I do Golden Gate assembly using a vector cut with regular type II restriction enzymes?
SnapGene 6.2 and later allows use of two non-type IIS enzymes as the vector input for a Golden Gate reaction with the following caveats:
- The overhang length and overhang recess for each enzyme matches those for the chosen Type IIS enzyme
- The selected Type IIS enzyme does not cut within the vector sequence
For example, the Type IIS enzyme BsaI generates a 3'-recessed, 4 nucleotide overhang. Enzymes such as NotI, BamHI, XmaI, NcoI, XbaI (and many others) can create overhangs compatible with overhangs generated by BsaI.
Open a Plasmid Vector with Suitable Sites for Cloning
Open a suitable vector sequence.
In this example we will used the vector-based XbaI and BamHI sites. These enzymes create 4 nucleotide 3'-recessed overhangs compatible with overhangs generated by BsaI.
Perform Golden Gate Assembly
Click Actions → Golden Gate Assemble → Insert Fragment or Insert Multiple Fragments to start the Golden Gate Assembly tool.
Define the Vector Region for Replacement
If a DNA file is open when you run this tool it will be automatically selected as the vector. If required, use the "Vector:" dropdown menu to choose an alternative vector file.
Select the Golden Gate (Type IIS Enzyme) to use.
Select the option to "Digest with other Enzymes".
Enter enzyme names in the fields provided, or use the dropdown menus to choose a pair of appropriate enzymes with unique overhangs compatible with your selected Golden Gate enzyme.
The selected enzymes will be displayed in Map and Sequence view, and the "Region to replace" will be selected.
Define the Insert Fragment
Switch to the Fragment tab, set the "Source of the Fragment:", then in Map or Sequence View, select the feature or region for the insert fragment.
If your Insert fragment sequence already has a pair of appropriate flanking Type IIS sites then these will be automatically detected and selected, and the option to "Digest with ..." will be selected.
If your insert fragment sequence does not have Type IIS sites then the option to "Run PCR and then digest with ..." will be selected.
Design Primers to Add Type IIS sites to the Insert Fragment via PCR
Switch to the Product tab and click Choose PCR Primers....
Set the desired primer melting temperature (Tm) and number of nucleotides to include upstream of the BsaI site, then click Choose Primers.
Name the New Primers
Switch to the Fragment tab, and give the primers appropriate names.
Validate the Product
Switch back to the Product tab, switch to Sequence view and confirm the vector/insert boundaries are correct.
Enter an appropriate name for the new product, then click Assemble to create the new product file.
SnapGene will warn that the overhangs are palindromic. Palindromic ends will reduce the predicted "Assembly fidelity" as they allow insert-insert ligation and vector-vector ligation, and consequently, a higher frequency of incorrect assembly.
View the Product History
View the product file History view so see a summary of the steps simulated for the Golden Gate Assembly.