How do I simulate NEBuilder Hifi DNA Assembly® in SnapGene?
In this lesson we will "clone" the mEGFP coding sequence (CDS) into the controlled expression vector pET-52B(+). The mEGFP CDS will be positioned in-frame with the vector-based N-terminal Strep-tag II and C-terminal Thrombin + 10xHIS-tag.
Open or Import a Vector Sequence
In this example the vector fragment generated by digestion with SmaI and SacI will be replaced by the insert in the NEBuilder® Hifi DNA Assembly reaction.
Import or open a sequence file for the plasmid vector that will be used for NEBuilder HiFi DNA Assembly®.
In Map view, or Sequence view, set the region for replacement by one of the following methods:
- Click within the sequence to select the insertion point, or
- Select a specific region that will be replaced by the insert, or
- Click on an enzyme site to select it as the insertion point, 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.
Start the NEBuilder HiFi DNA Assembly® Tool
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 insert.
If a vector sequence is not open when you start the NEBuilder Hi fi DNA Assembly® tool then you can choose and define the vector in the Vector tab.
Click Actions → NEBuilder HiFi DNA Assembly® → Insert Fragment....
The NEBuilder HiFi DNA Assembly® tool opens showing the "Vector" tab. The option to "Linearize with restriction enzymes" is automatically selected, and the appropriate vector restriction fragment for replacement selected.
If you want to use a different vector then click the "Vector:" dropdown to chose a new file, then set the "region for replacement".
If required, set the vector orientation using the "Orientation of Vector" buttons.
Specify the Insert Fragment Source Sequence
Switch to the "Fragment" tab.
There are a number of ways to specify the fragment source sequence, you can use any one of the following methods:
- Click the dropdown to set the "Source of fragment", or
- Click the "Click here" link to browse and set the "Source of Fragment".
- Drag and drop a sequence file into the Fragment panel.
- Copy a sequence from any compatible source file and paste to import it as the fragment source.
In Map or Sequence view, select the region of the fragment sequence to be inserted into the vector.
In Sequence view, locate the start of the insert and click to set the cursor to the start position, then scroll and shift click to set the end position.
In this example we will manually define the insert region. We will not include the mEGFP stop codon to allow in-frame fusion to the vector-based CDS encoding a C-terminal Thrombin cleavage site and 10xHIS-tag.
Design Primers to Amplify the Insert Fragment
In this example we have checked the option regenerate the partial vector-based SacI restriction site. This will allow use of the site in future experiments.
Switch to the Product tab and click Choose Overlapping PCR Primers.
Set the desired Tm for the primers.
Set the desired overlap length between vector and insert ends.
Set the target Tm for the NEBuilder HiFi DNA assembly®.
Click Choose Primers to design new PCR primers.
Validate the Vector/Insert Fusion Points
In this example we confirm that the vector-based "Strep-Tag II/HRV 3C" features are in-frame with the start of the mEGFP CDS.
In this example we confirm that the vector-based "Thrombin/10xHIS tag" features are in-frame with the end of the mEGFP CDS. We also confirm the SacI site is regenerated.
In the Product tab, switch to Sequence view. and check the fusion points at each end of the insert.
Name the New Primers and Make the Product Sequence
Switch to the Fragment tab.
Enter appropriate names for the new primers.
Enter an appropriate name for the new product.
Click Assemble to create the new NEBuilder Hifi DNA Assembly® product.
Click File → Save to save the new product sequence to an appropriate location on your computer.
View History
View the newly created product file, switch to History view to see all steps simulated during the NEBuilder Hifi DNA Assembly® procedure.
To show History colors highlighting the insertion, click the "Hifi Assembly" operation name.
Order the PCR primers
Seethe lesson on Exporting Primers for details on how to export and order the new primer sequences.