May 12, 2026

mtORF PCR

DOI
https://dx.doi.org/10.17504/protocols.io.q26g7oexkvwz/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.q26g7oexkvwz/v1
Protocol Citation: Jordan Sims, Francesca Cohn 2026. mtORF PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7oexkvwz/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 28, 2026
Last Modified: May 12, 2026
Protocol  Integer ID: 315907
Keywords: mtorf amplicon, mtorf, pcr, negative control active time, active time, total time
Abstract
Make mtORF amplicons from 79 samples and one negative control
Active time: ~1 hour
PCR time: ~2 hours
Total time: ~3 hours
Materials
Equipment required
  • Fine point Sharpie
  • 1.5mL tube rack(s)
  • PCR strip tube rack(s)
  • Insulated aluminum tube rack(s)
  • Vortex
  • Mini spinner with 1.5mL tube and PCR strip tube attachments
  • 1000µL pipette
  • 200µL pipette
  • 10µL pipette
  • Five miniPCR thermal cyclers (miniPCR, cat. #QP-1016-16)
  • Laptop with miniPCR software
 
Consumable materials (per sample)
  • 25µL Taq PCR Master Mix (Qiagen, cat. #201445)
  • 21µL molecular grade water
  • 1µL 0.1% BSA (FisherSci, cat. #FERB14)
  • 1µL 10µM mtORF forward primer
  • 1µL 10µM mtORF reverse primer
  • 1µL extracted DNA
  • One 10µL pipette tip
 
Consumable materials (per batch)
  • 5mL microcentrifuge tube
  • 10 PCR strip tubes
  • Four 1000µL pipette tips (for making master mix in Step 6)
  • Four 200µL pipette tips (for making and aliquoting master mix in Steps 6 & 8)
Remove 79 samples from the freezer to thaw on the bench
Remove one aliquot each of Taq PCR Master Mix, molecular grade water, 0.1% BSA, 10µM mtORF forward primer, and 10µM mtORF reverse primer from the freezer to thaw on the bench
Remove an insulated aluminum tube rack from the freezer and place PCR reagents into the rack once they are fully thawed
Prepare 10 sets of PCR strip tubes. Label strips with unique ID and mark which tube is first and which is last
Gently vortex all PCR reagents. Spin down in mini spinner, if necessary
Add PCR reagents to a 5mL microcentrifuge tube in the following volumes to prepare master mix:
  • 2050µL Taq PCR Master Mix (1025µL x 2)
  • 1722µL molecular grade water (861µL x 2)
  • 82µL 0.1% BSA
  • 82µL 10µM mtORF forward primer
  • 82µL 10µM mtORF reverse primer
Vortex the 5mL tube for 2 seconds
Aliquot 49µL master mix into all strip tubes. There should be some leftover at the end
Add 1µL extracted DNA to each PCR strip tube (or 1µL molecular grade water to negative control). Pipette onto the side of the strip tube to avoid introducing a bubble. Always double check that you are adding the correct sample to the correct strip tube
Spin the strip tubes down in mini spinner
Place strip tubes into the thermal cyclers. Record in the lab notebook which strips go into which thermal cycler
Plug in and power on thermal cyclers
Open the miniPCR software. Click the “Devices” tab, highlight the desired thermal cycler, and click “Connect” to connect to the thermal cycler
Click the “Library” tab, highlight the mtORF protocol, and select “Run”. Choose the appropriate thermal cycler, then select “Upload”.
Note: mtORF protocol is 94ºC for 60s; 40X 94ºC for 30s, 53ºC for 30s, and 72ºC for 75s; and 72ºC for 300s
Double check that the PCR protocol has started running
Repeat Steps 13–15 four more times to start the remaining PCR reactions
Store PCR amplicons in the fridge