Dependence of the ligation efficiency of large DNA fragments isolated from agarose gels on the purification method

Abstract

The isolation of large DNA fragments (> 5 kb) from agarose gels can be problematic. The DNA yield is often low, and the purification may be insufficient for subsequent reactions such as sequencing or ligation. Here we have compared a number of commonly used methods and commercial kits for DNA recovery. Large DNA fragments (12 and 14 kb, respectively) were isolated from agarose gel and purified, the recovery yield was tested, and a well-defined amount of purified DNA was used in a standard ligation reaction. We observed dramatic differences in the efficiency of this ligation reaction depending on which recovery method had been used. However, the respective ligation efficiencies of the different methods were not related to their recovery yields.