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.
#CREATE PRIMERS IN SERIAL CLONER HOW TO#
In this video, we will demonstrate how to use the NEB Golden Gate Assembly Tool. Tutorial for NEB Golden Gate Assembly Tool.
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