Effects of UV-induced DNA damage on vector ligation and transformation into bacterial cells / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To study the effects of UV irradiation on DNA ligation and transformation efficiency of the expression vector into competent bacterial cells.</p><p><b>METHODS</b>The expression vector was digested with the restriction enzyme SfiI, and the purified target DNA fragments were exposed to UV light at different wavelengths. Ligation and transformation experiments with the exposed fragments were carried out and the colony number and transformation efficiency were assessed.</p><p><b>RESULTS</b>The transformation efficiency of the DNA with a 5-min exposure to 302 nm UV was 60 colonies per nanogram of the DNA, as compared with 20400 for the DNA exposed to 365 nm UV. The time course experiment showed that prolonged DNA exposure to 365 nm UV light was associated with lowered transformation efficiency. DNA exposure for 30 min caused a reduction of the transformation efficiency to lower than 50% compared to that of DNA without UV exposure. But with a 15 min exposure, the DNA maintained a transformation efficiency more than 70%, which was sufficient for most molecular biology experiments.</p><p><b>CONCLUSION</b>In construction of the expression vector, it is advisable to prevent the target DNA from UV exposure. When UV exposure is essential, we suggest that 365 nm UV be used and the exposure time controlled within 15 min.</p>