The Addgene Blog

Master plasmid fundamentals, CRISPR techniques, AAV serotype selection, and antibody applications. Written by scientists, for scientists.

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Schematic of Fragmid assembly process from day one to day four and resulting vector architecture.  On day one fragment plasmids plus a destination vector are assembled using Golden Gate (BbsI). On day two the assembled vector containing a Guide (2xBsmBI), Promoter, N-terminus, Cas protein, C’terminus, and 2A-Selection undergoes an exonuclease V cleanup followed by transformation and plating. On day three two colonies per construct are picked, miniprepped and restriction digested for gel validation/whole plasmid sequencing. Below the assembled vector are examples of six vector architectures including pRDA_512 lentivirus, pRDA_722 lentivirus, pRDA_789 CROPseq lentivirus, pRDA_889 AAV, pRDA_575 Piggyback, and pRDA_791 empty plasmid.
Graphic representing the choices made in the technical design of a western blot.
Two flow plots with a giant population. One has a gate around the far right hand side of the population.
A graphic depiction of a serial dilution.
Steps of rAb production.
Seven interns standing behind their posters and smiling.

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