2.2.2 Prevention of TAat adapters’ self-ligation
The self-ligation of TAAT adapters was tested with restriction enzyme AseI(New England Biolabs (Beijing) LTD). This experiment had two groups: adapters without and with AseI. For T4 DNA ligase group, 200 ng of TAat adapters, 5 μL 10X T4 DNA ligase buffer, and 1 μL of T4 DNA ligase (400 uints) were added to a total of 50 μL reaction system. The composition of the AseI group was the same as that of the T4 ligase control group, except that 2.5 μL (25uints) of AseI restriction endonuclease was added. The reaction was carried out overnight in a 37°water bath. After the reaction was complete, 5 μL of the reaction products were electrophoresed for each group.
The combination of MseI and AseI was tested in promoting the ligation of insert and adapters in addition to the decrease of adapters’ self-ligation. Five groups were set up for this experiment: artificial template alone, template with MseI, the TAat adapter self-ligation group, template plus adapters with addition of MseI and T4 ligase, and template plus adapters with addition of MseI, T4 ligase and AseI group as summarized in Table 3.
The ligation products of the fifth group were amplified by PCR, and the PCR products were subjected to Sanger sequencing in Sangon Biotech. The primer pairs were with the sequences 5-ccatccagcatccaactcggtgggggtggggat-3, and 5-gtcagctccacgcatacattatacg-3. PCR reaction performed in a total volume of 20 μL system containing 2× Taq Master Mix 10μL, adapter primer 1μL, target fragment primer 1μL, ligation product 3μL. The PCR program consists of 95°C 2min 1cycle, 95°C 10s, 58°C 20s, 72°C 30s, 20cycles, and a final cycle at 72°C 1min.