Simulate NEBuilder HiFi DNA Assembly®

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 "clone" the eGFP coding sequence (CDS) in the controlled expression vector pET-52B(+).  The eGFP CDS will be positioned in-frame with the N-terminal vector-based  Strep-tag II and in-frame with the C-terminal vector-based 10xHIS-tag.

Summary of steps in this lesson:

1. Define the pET-52B(+) SmaI-SacI restriction fragment used for NEBuilder HiFi DNA assembly®.

2. Define the mEGFP insert fragment sequence for PCR amplification and subsequent NEBuilder HiFi DNA assembly®.

3. Have SnapGene design primers for PCR amplification of the insert with extensions to create overlap with the vector SmaI-SacI fragment.

4. Simulate the NEBuilder HiFi DNA assembly® reaction between the vector and overlapping insert fragment.

Open and Prepare a Cloning Vector Sequence

Import or open a sequence file for the plasmid vector that will be used for NEBuilder HiFi DNA assembly®.

In this example we will download the pET-52b(+) vector from the SnapGene "Online Sequence Library".

Open or Import a Vector Sequence

Open a vector sequence file, or to import a vector from the SnapGene Online Library click File → Import →  SnapGene Online Sequences.

Search for pET-52 to find the file, select the file in the list, and click Import to download and open the sequence as a new file.

Check the Vector Orientation

Switch to Sequence view and locate the multiple cloning site (MCS). In this example  vector, the MCS and expression elements (Strep Tag II signal peptide and HIS-tag) are in the reverse orientation.

Click menu View → Flip Sequence to reverse complement the pET-52b(+) vector sequence. This will simplify the cloning procedure.

Click File → Save to save the pET-52b(+) vector to an appropriate location on your computer.

The start codon and Strep-Tag II features are now shown in the forward direction.

Define the Vector Region for Replacement

Select the restriction fragment for replacement by the insert. In this example we select the SmaI site label, then shift click on the SacI site label to select the region for replacement.

Start the NEBuilder HiFi DNA Assembly® Tool

Click Actions → NEBuilder HiFi DNA Assembly® → Insert Fragment.

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.

Select an Insert Fragment

Switch to the "Fragment" tab, set the "Source of Fragment" from the dropdown menu, or click Browse to locate the insert sequence file on your computer.

In this example we will manually define the insert region, excluding the mEGFP stop codon, to allow in-frame fusion to the vector-based CDS encoding a C-terminal Thrombin cleavage site and 10xHIS-tag.

Switch to Sequence view, locate the start of the insert and click to set the cursor to the start position.

Scroll to the end of the insert and SHIFT-click to select the desired insert region.

Design Primers to Amplify the Insert Fragment

Switch to the Product tab and click Choose Overlapping PCR Primers.

Set the desired Tm for the primers.

Set the desired overlap range for vector and insert ends.

Set the target Tm for the NEBuilder HiFi DNA assembly®.

In this example we will choose to regenerate the vector SacI restriction site, check the option to regenerate the SacI site.

Click Choose Primers to design new PCR primers.

Validate the Vector/Insert Fusion Points

In the Product tab, switch to Sequence view. In this example we observe that the vector-based Strep-Tag II/HRV 3C site is in-frame with mEGFP.

Scroll to the end of the insert fragment, mEGFP is observed to be in-frame with the vector-based Thrombin/10xHIS tag features, so no further adjustment of the insert is required.

Note the SacI site has been regenerated.

Name the New Primers

Switch back to the Fragment tab.

Enter appropriate descriptive names for the two newly designed 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 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.

Order the PCR primers

Seethe lesson on Exporting Primers for details on how to export and order the new primer sequences.