How do I simulate circularization of a DNA fragment by NEBuilder® HiFi DNA Assembly?
You can circularize a single fragment by NEBuilder® Hifi DNA assembly in SnapGene 6.0 and later.
Open a Sequence
Open the sequence for recircularization by NEBuilder® HiFi DNA Assembly.
The source sequence can be a linear sequence, or a circular sequence.
In Map view or Sequence view, select the region of the sequence to be recircularized.
Alternatively, right click on the sequence and from the contextual menu click Select Range to specify a precise nucleotide range.
Alternatively, you can select the region to be removed, then click Edit → Invert Selection to select the region of the sequence to be recircularized.
Start the NEBuilder® Hifi DNA Assembly Tool
Click Actions → NEBuilder Hifi DNA Assembly → Assemble Multiple Fragments....
By default the "NEBuilder® Hifi DNA Assembly:Assemble Multiple Fragments" tool expects two input fragments.
The open document is set as "Fragment 1".
Click the "Number of Fragments" dropdown and choose "Fragment 2".
With "Fragment 2" selected, click the minus (-) button to delete the second fragment.
The selected region of the input sequence is now ready for primer design to create overlapping ends.
If your input sequence is a linear fragment that already has appropriate overlapping ends for Gibson Assembly, then:
- Choose the option "Use directly as the fragment".
- Give the Product an appropriate name.
- Skip the following "Design PCR primers step" and click Assemble.
Design PCR Primers
Switch to the Product tab and click Choose Overlapping PCR Primers....
Set the desired Tm for PCR, and Tm and overlap for assembly.
Check the option to "Generate a circular product".
Click Choose Primers to design appropriate primers for amplification of the fragment.
Name the Primers
Switch to the Fragment tab and enter appropriate names for the newly designed primers (Optional).
Create the Product Sequence
The length of the overlap created by PCR using the newly designed primers, and the overlap Tm, is marked in the bottom panel.
Enter an appropriate name for the product sequence and click Assemble to generate a new product sequence file.
View Product Sequence History
The product sequence History records the above procedure as "PCR and Hifi DNA" Assembly steps.