Protocol for generating break in phosphodiester backbone with Tth Argonaute (TtAgo, NEB #M0665)
- Set up reaction as follows:
COMPONENTS REACTION (20 µl) RATIO ssDNA OR dsDNA Up to 1 pmol 1 ThermoPol® Reaction Buffer (10X) (NEB #B9004) 2 µl — ssDNA Guide Oligonucleotide
1 µl (5 pmol) 5 Tth Argonaute (1 µM) 1 µl (1 pmol) 1 Nuclease-free water (NEB #B1500) to 20 µl —
- Incubate at 80°C for 30–60 minutes.
- Stop reaction by rapid cooling to 4°C; TtAgo is not active below 65°C.
- TtAgo can be degraded/inactivated (if necessary) by treatment with Proteinase K (NEB #P8107) or Thermolabile Proteinase K (NEB #P8111) prior to clean up by one of the following:
* Optimal ssDNA guide oligonucleotides are 16-18nt in length and must be 5’-phosphorylated (we recommend T4 Polynucleotide Kinase, NEB #M0201).