May 12, 2026

NlaIV Digestion

DOI
https://dx.doi.org/10.17504/protocols.io.81wgbjb43vpk/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.81wgbjb43vpk/v1
Protocol Citation: Jordan Sims, Francesca Cohn 2026. NlaIV Digestion. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbjb43vpk/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: 315909
Keywords: restriction enzyme active time, enzyme active time, nlaiv, minutes total time, total time, hour, active time, minute
Abstract
Digest 112 samples with NlaIV restriction enzyme
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 rCutSmart Buffer (NEB, cat. #B6004S)
  • 0.5µL stock NlaIV restriction enzyme (NEB, cat. #R0126S)
  • One 20µL pipette tip
 
Consumable materials (per batch)
  • One 1.5mL microcentrifuge tube
  • Two 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 NlaIV enzyme and rCutSmart Buffer from the freezer. Vortex gently to mix. Spin down in mini spinner, if necessary
Prepare NlaIV working solution by combining 62µL rCutSmart Buffer and 62µL stock NlaIV enzyme in a 1.5mL microcentrifuge tube
Aliquot 1µL NlaIV 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 65ºC for 20 minutes
If possible, visualize digests immediately on gel. Otherwise, store in fridge