Protocol for generating break in phosphodiester backbone with Tth Argonaute (TtAgo, NEB #M0665)

TtAgo is a DNA-guided endonuclease that will cut a complementary sequence between the bases corresponding to positions 10 and 11 of the guide oligonucleotide.*  The protocol below can be used for general applications, and can be scaled for larger or smaller reaction volumes. The most important item to consider when setting up a reaction is the molar ratio of the individual components. Guide oligonucleotide should always be kept in excess of TtAgo. For further details on how to design guides and setup reactions please see GUIDElines for optimization of Tth Argonaute (TtAgo) reactions, which contains details pertaining to guide design, reaction setup and protocol modifications.

  1. Set up reaction as follows:

    ssDNA OR dsDNA Up to 1 pmol 1
    ThermoPol® Reaction Buffer (10X) (NEB #B9004) 2 µl
    ssDNA Guide Oligonucleotide
    (5 µM)
    1 µl (5 pmol) 5
    Tth Argonaute (1 µM) 1 µl (1 pmol) 1
    Nuclease-free water (NEB #B1500) to 20 µl
  2. Incubate at 80°C for 30–60 minutes.

  3. Stop reaction by rapid cooling to 4°C; TtAgo is not active below 65°C.

  4. 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:

    1. Column purification (we recommend the Monarch® PCR & DNA Cleanup Kit (NEB #T1030)) or

    2. Agarose gel extraction (we recommend the Monarch Gel Extraction Kit, (NEB #T1020)), or

    3. Phenol/chloroform extraction followed by ethanol precipitation.


* Optimal ssDNA guide oligonucleotides are 16-18nt in length and must be 5’-phosphorylated (we recommend T4 Polynucleotide Kinase, NEB #M0201).