How do I do Golden Gate assembly using a vector cut with regular type II restriction enzymes?
SnapGene can use a vector linearized with two non-type IIS enzymes as the vector input for a Golden Gate reaction with the following caveats:
- Vector linearization is achieved using two enzymes that generate unique (non-complementary) overhangs
- 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 pET-52b(+)-GG vector. The enzyme sites BamHI and XbaI will be used for linearization. These enzymes create 4 nucleotide 3'-recessed overhangs compatible with overhangs generated by BsaI.
Linearize the Plasmid with Two Appropriate Enzymes
Click on the first enzyme site to select it, then shift click on the second enzyme site to select the fragment to be discarded during linearization.
If the wrong fragment is selected then reverse the site selection order.
In the above example, the BamHI site is selected first, the XbaI site second.
Click Actions → Linearize.
Enter an appropriate name for the new sequence, then click Linearize.
A new unsaved file will be created. Click File → Save to save the file to an appropriate location on your computer.
Perform Golden Gate with the Linearized Fragment
Click Actions → Golden Gate Assemble → Insert Fragment or Insert Multiple Fragments to start the Golden Gate Assembly tool.
The Golden Gate tool will automatically set the option to "Use directly as the Linearized Vector".
Use the dropdown to set the desired Type IIS enzyme to use for the Golden Gate reaction.
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.
Design Primers to Add BsaI sites to the Insert Fragment
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.
View the Product History
View the product file History view so see a summary of the steps simulated for the Golden Gate Assembly.