...pLKO.1 sequencing primer (5’ CAA GGC TGT TAG AGA GAT AAT TGG A 3’). TIP: You may need to adjust the sequencing...control Negative control vector containing non-hairpin insert. pLKO.1 – scramble shRNA Negative control...DNA concentration. C.4 Ligating and Transforming into Bacteria Use your ligation method of choice. For ...oligos C.3 Digesting pLKO.1 TRC-Cloning Vector C.4 Ligating and transforming into bacteria D. Screening for...AgeI. Forward and reverse oligos are annealed and ligated into the pLKO.1 vector, producing a final plasmid... When visualizing DNA fragments to be used for ligation, use only long-wavelength UV light. Short wavelength...For a standard T4 ligation, mix: 2 μL annealed oligo from step C.2 20 ng digested pLKO.1 TRC-cloning vector...