NEBUILDER® Assembly Tool 2.0 Fragments Amplified by PCR

This video demonstrates how the NEBuilder Assembly Tool can be used to generate overlap sequences for the assembly of two fragments into a vector. In this scenario, all fragments are amplified by PCR and the vector does not have convenient restriction sites.

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NEBuilder 2.0 fragments amplified by PCR. In this video, we will demonstrate how the NEBuilder Assembly Tool can be used to generate overlap sequences for the assembly of two fragments into a vector. In this scenario, all fragments are amplified by PCR. The vector does not have convenient restriction sites where we would like to clone the insert, so we will amplify the vector and generate a 50 base pair deletion in the vector sequence. Begin by clicking 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. Click new fragment. For this demonstration. We will use preloaded sample sequences. Note that all vectors available from NEB are preloaded in the tool. Click use sample sequences. Select a cloning vector from the dropdown menu, in this case pUC19. The selected pUC19 sequence is now loaded in the sequence file. 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 PCR. To generate a 50 base pair deletion between basis 100 and 151 of the vector, we'll begin by entering the start base of 151 and with the end base of 100. The entire vector will be amplified except for that 50 base pair fragment. The start bases coordinate 151, the most five-prime sequence in your PCR amplified vector. Note that the plasmid diagram on the right shows the region that will be amplified. If you prefer, you can use custom primers instead of having the tool design primers for you. In this case, click specify custom primers and enter the custom primers here. Now click add. This will take you to the next page where the first set of primers will appear. This is also the page that gives you the option of adding more fragments to the assembly. To add the first insert, click new fragment, then click use sample sequences. From the dropdown menu, select the insert. In this example, the LacZ full sequence. Because this is an insert fragment, do not select vector or circular. Name your second fragment. Select PCR as the method of production. Check the start and end bases. In this case, we are adding the entire sequence. Click add. On the next page you will see a simple map showing the assembly so far. Note that the insert 5' end is joined to base 100 and the 3' end is joined to base 151 in the vector. Let's add another fragment. Click new fragment. From the dropdown, we are going to select GFP as our third fragment. Name the fragment. Select PCR as the method of production. Check the start and end bases. In this case, we will be adding the entire sequence. Click add. On the next page, you will see that the map on the right now shows the construct containing the three selected fragments depicted in three different colors. The fragments can be rearranged by dragging and dropping the colored bars into the preferred order. You can also delete fragments or edit them by using the functionalities/icons shown in the colored bars. You can also click the bars to view 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. Now click done. On the next page there will be a summary of your assembly. The summary contains a simple map of the assembled product, a sequence and multiple formats that can be exported and a color-coded sequence so that you can easily identify the assembly junctions. The required oligo section shows the oligo sequence which can be shown in FASTA or IDT compatible sequence formats as well as the Tm and annealing temperature of those oligos. The lower case five prime end is the overlap region of the primer. It is color-coded to match the color of the adjacent fragment in the assembly. The upper case 3' end is the gene specific portion of the oligo that acts as the primer for PCR. It is also color coded to match the PCR template. 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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