[Effects of UV-induced DNA damage on vector ligation and transformation into bacterial cells].
OBJECTIVE
To study the effects of UV irradiation on DNA ligation and transformation efficiency of the expression vector into competent bacterial cells.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Huang
Wan-ling
WL
School of Pharmaceutical Sciences, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. lenomwanling@163.com
Li
Chang-zheng
CZ
Chen
Zhen-rui
ZR
He
Wei
W
Zhou
Ye
Y
Zhou
Zhi-gang
ZG
Liu
Shu-wen
SW
Zhou
Chen
C
D001419:Bacteria / Q000235:genetics*; D004249:DNA Damage / Q000528:radiation effects*; D004260:DNA Repair; D005822:Genetic Vectors / Q000528:radiation effects; D014169:Transformation, Bacterial / Q000528:radiation effects*; D014466:Ultraviolet Rays