DNA ligation of ultramicro volume using an EWOD microfluidic system with coplanar electrodes

An automatic ultramicro-volume DNA ligation process using a coplanar electrode type of electrowetting-on-dielectric (EWOD) microfluidic system was designed for economy of reagent. The droplets, containing DNA, a ligation enzyme and a multi-salt reaction buffer, served to complete the ligation using a developed EWOD system. Droplets of ultramicro volume (0.3 νL) were successfully generated from reservoirs between one plate with coplanar electrodes and another plate with a hydrophobic surface free of electrodes. In one successful cloning, the total usage of reagents in an ultramicro-volume EWOD chip was 2.1 νL; no volume was wasted, in comparison with 85% waste with the standard protocol and 80% waste with a free-cover coplanar EWOD chip. The results also show that the entire process was accomplished without damage to the chip surface and without biomaterial annulment. With a design consisting of electrode pathways in a flexible pattern and multiplex reservoirs, an EWOD digital microfluidic system with coplanar electrodes would be improved as an efficient parallel DNA-cloning system in the construction of an artificial library or expression library.