■ PAUSE POINT Purified PCR product can be stored at –20 °C overnight. 112Ligate the digested pUC19 backbone and PCR product at a 1:3 vector:insert ratio and incubate it at room temperature for 15 min. As always, it is important to include a vector-only ligation control. ComponentAmount (μl)Digested pUC19x (50 ng)Digested PCR product (insert)x (1:3 vector to insert molar ratio)T7 Ligase1Rapid Ligation buffer, 2×10ddH2OUp to 20Total volume20113Treat the ligation reaction with PlasmidSafe exonuclease to digest any residual linearized DNA. This step is optional but highly recommended. ComponentAmount (μl)Ligation reaction from Step 11211PlasmidSafe buffer, 10×1.5ATP, 10 mM1.5PlasmidSafe exonuclease1Total15114Transformation of bacteria. Transform the PlasmidSafe-treated plasmid into a competent E. coli strain, according to the protocol supplied with the cells. We recommend Stbl3 for quick transformation. Briefly, add 5 μl of the product from Step 113 into 20 μl of ice-cold chemically competent Stbl3 cells; incubate the mixture on ice for 10 min, heat-shock it at 42 °C for 30 s, return it immediately to ice for 2 min, add 100 μl of SOC medium and plate it onto LB plates containing 100
