TITLE: 12S PCR Metabarcoding Protocol for Fish Detection in Estuarine Samples
AUTHORS: Fouad El Baidouri, Heather L. Gilbert, Alison Watts

[DESCRIPTION]
This methodology has been developed for the use of MiFish-U and MiFish-E primers multiplexed with Platinum SuperFi II (2X) and Zymo clean up under a touchdown PCR program, aimed at the specific amplification of fish species from estuarine samples with inhibitors. The primers, originally developed by Miya et al., 2015, and Kawato et al., 2021, target the hypervariable region of the mitochondrial DNA 12S rRNA gene in fish species including Elasmobranchii. Platinum SuperFi II (2X) is a high fidelity polymerase and is well suited for multiplexing as it can work using primers with different melting temperatures at 60°C.

[GUIDELINES]
Before preparing the PCR mix and adding DNA, thoroughly clean all work surfaces with a 5%-10% bleach solution, followed by distilled water (dH2O). Ensure that surfaces are cleaned both before and after use. Always wear lab coats, gloves, and change them when moving between different work areas to prevent cross contamination. Clean pipettes regularly using a 5%-10% bleach solution, followed by a rinse with dH2O. Dispose of all hazardous waste in designated disposal areas to maintain a safe and orderly laboratory environment.

[STEPS]
SECTION: DNA clean up using Zymo
1. Before setting up the PCR mix, purify the DNA using the Zymo OneStep PCR Inhibitor Removal Kit as indicated by the manufacturer (CAT: D6030 or D6035). After the clean up, dilute the DNA at a 1:5 ratio. Follow the the Guidelines & Warnings for lab space preparation and clean up. See Description above for a general overview of this protocol.
SECTION: Prepare the primer mix for multiplexing
2. Prepare an equimolar mix of MiFish-U-F and MiFish-E-F (with TruSeq or Nextera illumina adapters) of the Forward primers and an equimolar mix of the Reverse primers (final concentration for each primer in the PCR mix is 5 μM). See the Materials section above for details.

Prepare the Forward primer mix with equimolar concentration (final 5 μM)

Prepare a combined Forward primer mix:
- 90 μL of PCR grade water 
- Add 5 μL of MiFish-U-F stock solution (100 μM)
- Add 5 μL of MiFish-E-F stock solution (100 μM)
This results in a mix containing both Forward primers at a final concentration of 5 μM each.

Prepare a combined Reverse primer mix:
- 90 μL of PCR-grade water 
- Add 5 μL of MiFish-U-R stock solution (100 μM)
- Add 5 μL of MiFish-E-R stock solution (100 μM)
This results in a mix containing both Reverse primers at a final concentration of 5 μM each.
SECTION: Prepare PCR reagents
3. IMPORTANT: The PCR is run in triplicate for each sample and the products are pooled together before running the E-Gel and preparing the Library.

Set up the PCR reaction for a total reaction volume of 20 μl by mixing the following components (amounts are per sample):

PCR grade H2O: 2 μl
Platinum SUPERFI II Master Mix (2X): 10 μl
Forward primer mix (5 μM): 2 μl
Reverse primer mix (5 μM): 2 μl

Aliquot 16 μL of the mixture into each well to be used in the 96 well PCR plate
Add 4 μL of DNA (Zymo cleaned and diluted at a 1:5 ratio)
Total Volume: 20 μl
SECTION: Set up Touchdown program on thermocycler
4. We use a touchdown protocol by setting up an initial annealing temperature of 69.5°C to increase the specificity for the target.


 

SECTION: PCR check
5. After amplification, check the success of the PCR reaction by loading the sample onto a 2% agarose gel using a 1:3 dilution of the products and a 100 bp ladder (diluted 1:10).
NOTE: In the same lane, the gel should show one main band slightly below 300 bp. In some cases another band will appear (mainly off-target) at slightly below 400 bp.