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.