May 12, 2026

AciI/BseYI Digestion

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvjk4pxgk5/v1
  • Jordan Sims1,
  • Francesca Cohn1,2
  • 1UC Berkeley;
  • 2Stanford University
  • Cryptic Pocillopora FP
Jordan Sims
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvjk4pxgk5/v1
Protocol Citation: Jordan Sims, Francesca Cohn 2026. AciI/BseYI Digestion. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjk4pxgk5/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 29, 2026
Last Modified: May 12, 2026
Protocol  Integer ID: 315915
Keywords: bseyi digestion digest, acil, bseyi, total time, hour, minutes total time
Abstract
Digest 112 samples with Acil and BseYI restriction enzymes
Active time: ~1 hour
Incubation time: 1 hour, 20 minutes
Total time: ~2 hours, 20 minutes
Materials
Equipment required
  • Heat block(s) with 112 slots for 0.5mL tubes
  • Fine point Sharpie
  • 1.5mL tube rack(s)
  • PCR strip tube rack(s)
  • Vortex
  • Mini spinner with 1.5mL tube attachment
  • 200µL pipette
  • 20µL pipette
  • 10µL pipette
 
Consumable materials (per sample)
  • One 0.5mL microcentrifuge tube
  • 0.5µL NEBuffer r3.1 (NEB, cat. #B6003S)
  • 0.5µL stock Acil enzyme (NEB, cat. #R0551S)
  • 0.5µL stock BseYI enzyme (NEB, cat. #R0635S)
  • One 20µL pipette tip
 
Consumable materials (per batch)
  • One 1.5mL microcentrifuge tube
  • Three 200µL pipette tips (for preparing working solution in Step 4)
  • One 10µL pipette tip (for aliquoting working solution in Step 5)
Turn on heat block and set to 37ºC
Label clean 0.5mL microcentrifuge tubes with SampleID by writing on the tube lid directly with a fine point Sharpie
Remove one aliquot each of stock Acil enzyme, stock BseYI enzyme, and NEBuffer r3.1 from the freezer. Vortex gently to mix. Spin down in mini spinner, if necessary
Prepare Acil/BseYI working solution by combining 62µL NEBuffer r3.1, 62µL stock Acil enzyme, and 62µL stock BseYI enzyme in a 1.5mL microcentrifuge tube
Aliquot 1.5µL Acil/BseYI working solution into the bottom of each labeled 0.5mL microcentrifuge tube
Transfer 5µL mtORF amplicons to each 0.5mL tube. Always double check that you are adding the correct sample to the correct tube
Flick each tube gently to mix. Spin down tubes in mini spinner
Incubate all tubes at 37ºC for 60 minutes, then 80ºC for 20 minutes
If possible, visualize digests immediately on gel. Otherwise, store in fridge