NEBUILDER® Assembly Tool 2.0 Restriction Enzyme Digest

This video demonstrates how to use the NEBuilder® Assembly Tool to build a construct using a restriction enzyme digested vector and two PCR-generated inserts.  The tool will help to design PCR primers containing the required overlap sequences.  We will also regenerate one of the restriction enzyme recognition sites.

Script

In this video, we will demonstrate how to use the NEBuilder Assembly Tool to build a construct using a restriction enzyme digested vector and two PCR-generated inserts. The tool will help to design PCR primers containing the required overlap sequences. We will also regenerate one of the restriction enzyme recognition sites. Begin by clicking on the Settings tab. Choose the assembly product and the PCR polymerase. The assembly can be linear or circular. We will be making a circular assembly product. Click Done. Now, we will add the fragments to the assembly. Begin by clicking on Build. Then, click New Fragment. For this demonstration, we will use preloaded sample sequences. Note that all vectors available from NEB are preloaded into the tool. Click Use Sample Sequences. Select a cloning vector from the dropdown menu. In this case, pUC 19. The select a sequence is now loaded in the sequence field. Make sure that you check both vector and circular. In step two, name your fragment. In step three, select a method of production. In this example, we are using a fragment produced by restriction enzyme digestion. Now, select the restriction enzymes that you will use to digest the plasmid from the dropdown menu. We have chosen HindIII and EcoRI. Note that only restriction enzymes with one or two sites in the plasmid are available. By convention, the insert fragment will be cloned in the clockwise direction with the 5' end of the insert joined to the 5' end of the vector and the 3' end of the insert joined to the 3' end of the vector. In this case, the upstream 5' site is EcoRI, and the downstream 3' site is HindIII. Once you have selected the restriction enzyme sites, the map in the upper right will show the digested plasmid. You can use the Swap button to change the upstream and downstream sites. Notice how the plasmid map changes when the restriction enzyme sites are swapped. Click Add. On the next page, you can edit the fragment. Click the pencil icon to edit the fragment's properties or add a spacer. We will edit the fragment by regenerating the HindIII site. Site regeneration adds bases to primers in order to rebuild the restriction enzyme recognition site. We're not using primers to generate this fragment, but the information will be used in the design of the primers for adjacent fragments. Go to the downstream 3' site, and, under Site Regeneration, choose Strict. Strict regeneration adds all the bases required to regenerate the restriction enzyme site with no regard for the sequence of the adjacent fragment in the assembly. Minimum regeneration takes into account the sequence of the adjacent fragment in the assembly and will use bases from that fragment if appropriate to regenerate the restriction site. Click Update to make the changes in the fragment. Next, we will add insert fragments. Click Plus New Fragment. We will use a sample sequence. Select lacZ Full from the dropdown menu. Name the fragment. Select PCR as production method. Be sure that Autogenerate Primers is selected. If you prefer, you can use custom primers instead of having the tool design primers for you. Enter the start and end bases. In this case, we will use the entire sequence. Click Add. On the next screen, we will see a simple map of the plasmid vector and the lacZ insert. The 5' end of the lacZ insert is joined to the EcoRI site. The 3' end of the lacZ insert is joined to the HindIII site. Scroll down to see the required oligonucleotides. The sequence in black indicates the additional sequence added to regenerate the HindIII site. Now, let's add another fragment. Click Plus New Fragment. We will use another sample sequence. Select GFP from the dropdown menu. Name the fragment. Select PCR as the production method. Be sure that Autogenerate Primers is selected. Enter the start and end bases. In this case, we will use the entire sequence. Click Add. On the next screen, we see a simple map of the plasmid vector and the lacZ and GFP inserts. The 5' end of the lacZ insert is joined to the EcoRI site. The 3' end of the lacZ insert is joined to the 5' end of GFP, and the 3' end of GFP is joined to the HindIII site. Fragments can be rearranged by dragging and dropping the colored bars on the left into the preferred order. You can also delete fragments or edit them by using the functionalities, icons shown in the colored bars. Click the bars themselves to view the sequence of the assembly junctions, primer sequences, and the amino acid sequence for all six reading frames. The Notes section provides information about the characteristics of the primers. The Build settings shows the default settings used to generate the primers. Use this to change the assembly product, minimum overlap size, or polymerase used for PCR. Scroll down to the Required Oligos field. Each fragment generated by PCR has a pair of oligos. The 5' end of each oligo, seen in lowercase, contains the overlap sequence for the adjacent fragment in the assembly. The 3' end of each oligo, seen in uppercase, contains the PCR primer portion of the oligo. The GFP reverse primer also contains the sequence AGCTT, which are the bases added to the primer to restore the HindIII site. The TM for each primer and the predicted annealing temperature are all shown. The annealing temperature is calculated for the specific polymerase chosen in the first page. Click Done. On the next page, there will be a summary of your assembly. This summary page contains information on the fragments, the oligos required for PCR, which can be shown in faster or IDT-compatible sequence formats, as well as the TM and annealing temperature of those oligos. There is also a simple map of the assembled product, a sequence in multiple formats that can be exported and a color-coded sequence that you can easily identify the assembly junctions. Use the Load Save tab to save your assembly. Enter the name of the assembly in the Save Project field and export the file to your computer. To start a new assembly, click Build, and then start over. Click Okay to delete the existing project, and then you can start your next assembly.
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