template
HpaII
CGat adapter
CGat adapter+template+HpaII
CGat adapter +template+HpaII+ClaI
template 200ng 200ng 0 200ng 200ng
CGatadapter 0 0 300ng 300ng 300ng
10X CutSmart™ Buffer 5 5 5 5 5
ATP(10μM) 5 5 5 5 5
T4DNA Ligase 0 0 1 1 1
HpaII 0 1.5 0 1.5 1.5
ClaI 0 0 0 0 2.5
Total volume 50 50 50 50 50
Reacted overnight at 37 °C, T4 ligase was inactivated at 65 °C for 20 min Reacted overnight at 37 °C, T4 ligase was inactivated at 65 °C for 20 min Reacted overnight at 37 °C, T4 ligase was inactivated at 65 °C for 20 min Reacted overnight at 37 °C, T4 ligase was inactivated at 65 °C for 20 min Reacted overnight at 37 °C, T4 ligase was inactivated at 65 °C for 20 min Reacted overnight at 37 °C, T4 ligase was inactivated at 65 °C for 20 min