High-throughput protocol for phosphorylation of guide oligonucleotides in 96-well PCR plate format (NEB #M0665)

If a large number of guides are required, unmodified oligos may be ordered in 96-well PCR plate format. Many suppliers (e.g., Integrated DNA Technologies, Inc.) offer a 96-well PCR plate format picomole scale synthesis and will ship the oligonucleotides wet (be sure to request nuclease-free water rather than a buffered option). This format can greatly streamline the phosphorylation protocol. The following example protocol assumes a 200 pmol scale synthesis with 0.2 nmol normalized yield, shipped “wet” in nuclease-free water at a concentration of 10 µM (i.e., a final volume of 20 µl).

  1. Setup a reaction for each unmodified oligonucleotide to be used as a guide with argonaute. This can be done in PCR tube strips for convenience. For 40 µl reactions yielding 5µM 5′-phosphorylated guide, prepare the following in the order provided:
    DNA Oligonucleotide (10 µM) 20 µl (already on plate)
    Nuclease-free water (NEB #B1500) 15 µl
    T4 DNA Ligase Reaction Buffer (NEB #B0202) 4 µl
    T4 Polynucleotide Kinase (NEB #M0201) (100 u/µl) 1 µl
  2. Note: Volumes may need to be adjusted depending on the actual scale of the synthesis ordered.

  3. Incubate reactions at 37°C (phosphorylation), followed by 65°C for 20 min (heat-inactivate/denature T4 PNK).

  4. The denatured PNK does not interfere with the argonaute endonuclease reaction, and the guides are ready to use.

  5. With proper/adequate sealing of the plate, phosphorylated guides may be stored at 4°C for up to two weeks. If stored frozen at -20°C phosphorylated guides exhibit the same level of activity for several months to a year.