How do I simulate NEBuilder® Hifi DNA Assembly in SnapGene?
NEBuilder® Hifi DNA Assembly is a registered trademark of New England Biolabs Inc.
For information about NEBuilder Hifi DNA Assembly® see this link.
In this lesson we will simulate directional in-frame fusion of the SEC13 coding sequence (CDS) and EGFP CDS into the Pichia pastoris expression vector pIB2.
Open a Vector Sequence
Import or Open the vector sequence.
In Map or Sequence view:
- Click within the sequence to select the insertion point, or
- Click on an enzyme site to select it as the insertion point, or
- Select a specific region that will be replaced by the insert, or
- Select a restriction fragment that will be replaced by the insert.
To select a restriction fragment for replacement, click on the first restriction site, then shift click on the second restriction site. Alternatively, click on the first site then drag to the second site.
In the above example, vector digested with KpnI and SpeI will be used in the NEBuilder® Hifi DNA Assembly reaction.
Start the NEBuilder® HiFi DNA Assembly Tool
Click Actions → NEBuilder® Hifi DNA Assembly → Insert Multiple Fragments....
When starting the NEBuilder® Hifi DNA Assembly tool, the DNA sequence selection in the frontmost window will automatically be set as the vector region to be replaced by the inserts.
If a vector sequence is not open when you start the NEBuilder Hif DNA Assembly® tool then you can choose and define the vector in the Vector tab.
The NEBuilder® Hifi DNA Assembly tool opens showing the "Vector" tab, with the option to "Linearize with restriction enzymes" automatically selected and the appropriate fragment for replacement selected.
If required, set the vector orientation using the "Orientation of Vector" buttons.
Set the Number of Fragments for Insertion
By default the Ligate Fragments tool starts expecting two insert fragments.
Switch to the "Fragments" tab.
Click the +/- buttons to add or remove fragments. The number of fragments is displayed in the Tab Header.
For larger numbers of fragments, click the dropdown and choose "Number of Fragments". Enter the number of fragments and click OK.
Define the Insert Fragments Sequences
To define the inserts use in combination of the following methods:
- Drag and drop files into the "Fragments" panel.
- Set the "Fragment" via the dropdown then click the "Source of Fragment" dropdown and choose a file from the list of Open or Recent files, or click Browse to choose a file.
- Click the "Click here" links to browse and choose one or more files.
Reorder the Fragments Sequences
If fragments are in the wrong order, click the dropdown and choose "Order of Fragments".
Select one or more sequences and use the arrows to change the order, then click OK.
Define the First Fragment Region
In the Fragments tab, if required, use the dropdown to select "Fragment 1".
In Map view select the feature defining the fragment 1 insert, or alternatively, switch to Sequence view and manually select a precise insert region.
In the above example we plan to generate an in-frame fusion between the SEC12 CDS and the EGFP CDS. Therefore, we switch to Sequence view and select the precise SEC13 sequence region, excluding the SEC13 stop codon.
The option to "Use as a template for PCR" will be automatically set as SnapGene assumes the need to amplify the selection by PCR.
Define the Second Fragment Region
Use the dropdown to select "Fragment 2".
In the Fragments tab, in Map view, select the Feature defining the Fragment 2 insert region, or switch to Sequence view and manually select the insert region.
In this example, we click on the EGFP CDS feature to select it as the second fragment.
Design Primers to Amplify the Insert Fragments
Switch to the Product tab and click Choose Overlapping PCR Primers....
Set the desired Target Tm for the PCR primers.
Set the desired overlap length and the target Tm for the overlap regions.
Click Choose Primers to design PCR primers for amplification of the inserts.
If required, check on one or both options to either "Regenerate" or "Remove" the partial restriction sites at the ends of the vector.
Confirm the Vector/Insert Fusion Points
In the Product tab, switch to Sequence view. Scroll and check the fusion boundaries between each fragment.
In this example we confirm that the SEC13 CDS is in-frame with the EGFP CDS.
Name the New Primers and Create the Product Sequence
In the Fragments tab, click the dropdown and switch to each Fragment in turn, set appropriate names for each newly designed primer.
Set an appropriate name for the Product sequence.
Click Assemble to create the Product sequence.
Click File → Save to save the Product sequence to an appropriate location on your computer.
Switch to History view to see all steps simulated and all sequences/primers created during the NEBuilder® HiFi DNA Assembly procedure.
Order the PCR primers
See Export Primers for details on how to export and order the new primer sequences.