Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

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Two graphs, of fluorescence versus time and the residual (F - F_fit)/F_fit versus time, with vertical bands alternating gray and white every 5 seconds. The signal follows a prominent sawtooth pattern, decreasing during the gray period and recovering during the white period. Bottom panel (residuals) indicates ΔF/F reaches 75% and decreases slightly each cycle, but still reaches over 50% after seven cycles.
Exposed membranes showing a protein gradient with a primary antibody at two different dilutions.
schematic summarizing antibody validation approach for flow cytometry.
Side by side comparison of different antibody fragments relative to a full antibody. From left to right, Full IgG Antibody, F(ab’)2 Fragment, Fab’ Fragment, Fab Fragment, and Fv (scFv).
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.

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