This will bring up a color coded listing of the insert with both its upstream and downstream regions. Note that further information about junctions is available by selecting any insert in the graphic. Is the “Estimated ligation fidelity” high enough to ensure assembly success? If not, click on “Optimize” or “Refresh” to receive additional recommendations. For more details on warnings, please refer to the “Help” section of the tool. These include self-annealing primers that could generate a Golden Gate active false insert during PCR, an overhang that our T4 DNA ligase fidelity research identifies as leading to poor assembly efficiency, or a discontinuity or lack of a proper overhang between assembly sequences. Note the strongest warnings indicate issues that could lead to assembly failure. Check the “Notes” section for any warnings that should be assessed before moving forward. Are the inserts in the desired order? If not, rearrange an insert by selecting it and dragging it to its new position. At this point, you can review your planned assembly. You have now indicated your chosen destination plasmid vector backbone and the five inserts in the desired order. After all inserts are added, the graphic shows the pGGAselect vector backbone and all inserts. With each addition, the graphic will show the added insert on the map with the order of inserts shown above. In this demonstration, we will add inserts two, three, four, and five.Īlways remember to click on “Add” for each added insert. Repeat the steps above for each fragment you would like to add. Add the second, third, fourth, and fifth inserts in a similar way. This button will be disabled if the program detects an internal site in your insert sequence. As you can see, the graphic now shows both the pGGAselect vector backbone and the first insert. For our demonstration, however, we will use the entire sample insert sequences in the appropriate orientation as shown. If your insert sequence is part of an uploaded plasmid construct, it is important to identify the uploaded sequence as being circular in case your insert spans the beginning of the presumed linear sequence file. You can also generate the reverse complement if this is required for the correct orientation of your building assembly.
If loading your own insert sequence, you have the option of designating the beginning and end of the desired insert sequence by entering the “Start” and “End base” numbers from your uploaded sequence. Next, select “PCR-generated BsmBI-v2 sites” to automatically build flanking BsmBI sites in the primer designed for your insert PCR. Notice the vector and circular identifiers have not been checked as the entire sequence in the sequence window should be the desired insert. The sequence will now appear in the parsed sequence window and the insert name will be shown. Select “Insert 1” from the sample inserts dropdown menu. The graphic should now show the pGGAselect vector backbone. For other proposed destination plasmids, the tool will warn you if sites are not present or if they are improperly oriented. Since pGGAselect already has designed flanking BsaI, BsmBI, and BbsI sites at the assembly site of the plasmid. Next, select “Existing BsmBI-v2 cut sites”. Note the destination vector has been identified as your vector and as circular. You will also have the option of using the sequence or converting it to its reverse complement to fit in the correct orientation in your desired final assembly. If you are loading your own destination vector sequence, you can paste it in manually or load your GenBank, FASTA, or raw sequence file. The sequence will appear in the parsed sequence window and the vector name will be shown. Then select “pGGAselect” from the destination vector's dropdown menu. Add the destination vector by clicking on “Add Sequence”. In our example, we will select “BsmBI-v2” and use sequences that have no internal sites.Īdd the destination vector. If this is not possible, view our video on Golden Gate Domestication for strategies dealing with internal sites. Choose the restriction enzyme that has no internal sites in the DNA sequences to be used in the assembly. Enzymes available from NEB are shown in red. The tool will help design PCR primers to make amplicon inserts, check sequences for internal Type IIS restriction enzyme cut sites, and generate a report that shows the assembly graphically, and includes a full construct sequence file.īegin by selecting the Type IIS restriction enzyme you have chosen for your assembly design from the dropdown menu.
As an example, we will build an assembly from five sample inserts or modules using the pGGAselect destination plasmid supplied with our Golden Gate Assembly Kits. In this video, we will demonstrate how to use the NEB Golden Gate Assembly Tool. Tutorial for NEB Golden Gate Assembly Tool.
0 kommentar(er)
