Protocol for a substrate with 5-hydroxymethyluridine such as bacteriophage SP8 genomic DNA (M0659)
1. Phosphorylation of a substrate with 5-hydroxymethyluridine such as bacteriophage SP8 genomic DNA
- Prepare a 20 µl reaction as follows
DNA up to 1 µg T4 DNA Ligase Buffer (10X) 2 µl 5-HMUDK enzyme (20 units) 1 µl Nuclease-free H2O to 20 µl
- Incubate at 37°C for 30 minutes
2. Confirmation of phosphorylation of DNA by 5-HMUDK
- Prepare a NcoI-HF® digestion mix as follows:
|
DNA |
up to 1 µg |
| CutSmart Buffer (10X) | 2 µl |
| NcoI-HF (20U/µl) | 1 µl |
| Nuclease-free H2O |
to 20 µl |
2. Incubate for 30 minutes at 37°C.
3. Stop reaction by adding 10 µl of Gel Loading Dye, Purple (6X) (NEB #B7024)
4. Analyze by gel electrophoresis.
*Scale larger reaction volumes proportionally.
