The ratio of padlock DNA: primer is important to synthesize circular DNA template for subsequent rolling circle amplification. To optimize the synthesis condition, the volume of primer solution (10 μM) and padlock DNA solution (10 μM) was varied before T-4 DNA ligase was added to form the circular DNA. In Fig. , the gel image shows that when the padlock DNA: primer ratio changed from 2:1 to 1:2, the circular DNA band became the darkest accompanied by disappearance of the padlock DNA band. The other DNA bands were less affected. Therefore, we choose the padlock DNA: primer ratio of 1: 2 to prepare the circular DNA template in the remaining experiments.