High-throughput protocol for phosphorylation of guide oligonucleotides in 96-well PCR plate format (NEB #M0665)
- 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
Note: Volumes may need to be adjusted depending on the actual scale of the synthesis ordered.
- Incubate reactions at 37°C (phosphorylation), followed by 65°C for 20 min (heat-inactivate/denature T4 PNK).
- The denatured PNK does not interfere with the argonaute endonuclease reaction, and the guides are ready to use.
- 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.