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

  1. Prepare a 20 µl reaction as follows

    DNA up to 1 µg
    T4 DNA Ligase Buffer (10X) 2 µl
    5-HTDK enzyme (20 units) 1 µl
    Nuclease-free H2O to 20 µl


  2. Incubate at 37°C for 30 minutes

2. Confirmation of phosphorylation of DNA by 5-HMUDK

  1. 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.