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. 2022 Dec 21;5:1393. doi: 10.1038/s42003-022-04363-z

Table 2.

Circularization reaction ingredients for 6.0 μg of input dsDNA in 50 μl and cycler schedule.

Reagents
Component Volume (μl)
dsDNA, concentration ≥180 ng/μl per concentration to 6.0 μg
T4 DNA Ligase Buffer (10X B0202S) 5.0
T4 DNA Ligase (NEB M0202L) 3.0
Bsai-HF2 (NEB R3733L) 3.0
ATP (10mM NEB P0756S) 5.0
Nuclease-free H2O (Invitrogen 10977015) as needed to 50.0 μl
Cycler schedule
Step Temperature Time
Circularization 37 oC 60 min
T4 DNA ligase inactivation 65 oC 15 min
Pause 4 oC indefinitely