In a typical ligation reaction, a total of 100 ng of vectorwas usedwhereas;theconcentration of insert varies from 300 to 500 ng. For10μlreaction volume, 1μlligation buffer (provided by the manufacturer) and 0.5μlof T4-DNA ligasewere added in vector-insertmixture. The reaction was maintainedat 16oC for 14-16h.After ligation, 2μl of the ligation mixture (of total volume of10 μl reaction) was added to a vial of ultra-competentDH5α bacterial cells and incubated in ice for 30 minutes. The ligation mixture was allowed toheat shock at 42oC for 90 seconds followed by quick transfer onice. About1ml of LB broth was added to the tube and incubated at 37oC for 1 hour. The bacterial cells were then pelleted by centrifugation at 6000rpm for 5 min and plated on LB plate containing appropriate antibiotic

of ice cold deionized water (water was kept in the cold room overnight) was added to a 250 mL volumetric flask which was placed on ice. To the water, 52.1 mL of triethylamine solution (~7.2 M, Cat. No-T088, Sigma-Aldrich) was added with the help of a glass measuring cylinder, and mixed well. The volume of the solution was made up with ice cold water till the mark on the volumetric flask, and the flask was mixed well. A pH meter (Eutech instruments 510) was calibrated using pH 7.0 and pH 10.0 solutions (Eutech instruments 510). Approximately 100 mL of 1.5 M triethylamine solution from the volumetric flask was added to a 250 mL conical flask that was placed on ice. A magenetic bead was placed in the flask and the ice bucket was placed on the magnetic stirrer. The pH measuring probe was immersed into the solution, and CO2was bubbled through the triethylamine solution and stirred until the pH reached 8.5. The conical flask was covered with paraffin film and kept on ice until the solution wasused (within 1-4 h)

During the HPLC run,a triethylammonium bicarbonatesolution(1.5 M) was prepared. Triethylamine and water mixture generates heat, and therefore, approximately 100 mL