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322 Thibodeau-Beganny and JoungFig. 2. Structure and sequence of the reporter plasmid vector pKJ1712. A schematicof reporter plasmid pKJ1712 is shown. The region of the plasmid into which targetDNA-binding sites are cloned is represented as a black rectangle with correspondingdetailed sequence shown. Digestion of pKJ1712 with SapI Type IIS restriction enzymereleases a 45-bp fragment. Further treatment of the SapI-digested vector backbone withPfu DNA polymerase in the presence of dCTP nucleotide results in formation of theDNA overhangs illustratedc. 20 µL 10X Annealing buffer.d. 178 µL H 2O.e. Incubate at 95°C for 2 min, then shut off heat block and let tubes slowly coolto 35°C and then place on ice or 95°C for 2 min → –1°C/min → 25°C → 4°Cin a thermocycler. Store at –80°C.6. Ligate the purified pKJ1712 vector backbone to the annealed oligonucleotidebinding site as follows:a. 2 µL Purified SapI-digested, Pfu-treated pKJ1712 vector.b. 8 µL Annealed binding site oligos.c. 10 µL 2X Quick ligase buffer (NEB).d. 1 µL T4 DNA ligase (400 U/µL) (NEB).