NEB, M0648L, APOBEC3A

[{"group": "General", "name": "Materials Required but not Supplied dC- or 5mC-containing ssDNA Formamide (for dsDNA denaturation if needed) Microcentrifuge tubes Thermocycler or heat block Unit Definition One unit is defined as the amount of enzyme required to deaminate 5 pmol of a fluorescently labeled 44-mer oligonucleotide containing a single cytosine base in 30 minutes at 37°C in a total reaction volume of 20 μl in 1X Deamination Reaction buffer. Storage Temperature -20°C Reaction Conditions 1X Deamination Reaction Buffer Incubate at 37°C Heat Inactivation 70°C for 10 minutes Unit Assay Conditions 5 pmol of fluorescently labeled 44-mer oligonucleotide containing a single cytosine is incubated with 1 unit of APOBEC3A for 30 minutes at 37°C in a total reaction volume of 20 μl in 1X Deamination Reaction Buffer. The enzyme generated uracil-containing ssDNA is then incubated with USER enzyme in 1X rCutSmart Buffer to cleave the phosphodiester backbone at the uracil site. The resulting product is measured by capillary electrophoresis to determine APOBEC3A deamination activity.", "value": ""}]