RGG metabarcoding protocol for the 12S marker

Research Group Genetics Authors: Owen S. Wangensteen Mads Reinholdt Jensen Kim Præbel (kim.prae[email protected]) Norwegian College for Fishery Science Research Group for Genetics VOctober 2025 (MRJ) DNA AMPLIFICATION FOR METABARCODING This setup is for sequencing on the MiSeq/Novaseq with 12s primers using AmpliTag Gold MasterMix. 1) If you have extracted your samples in the eDNA Extraction lab, you bring your aliquots with you in two bags into the PCR-setup lab. Bring also your suit from the extraction lab. 2) Throw away the outer bag and clean with bleach the inner bag around your samples. 3) Put your samples in the fridge while you clean the hoods and equipment following the protocol. 4) Take necessary chemicals out of the freezer 30 mins prior to work to defrost. This takes approx. the same time as UV’ing the hood after cleaning, so do this simultaneously. 5) The PCR-mix is as follows: REAGENT VOLUME (µL) AmpliTag Gold Master Mix 10.00 Bovine serum albumin (BSA) 0.16 H2O 4.84 Primers Mix (F + R) 2.0 DNA template 3.0 6) Prepare the MasterMix for your samples in triplicates: MASTERMIX X1, µL X8, µL X100, µL (96-WELL PLATE) X?, µL AmpliTag Gold 3x10 = 30 240 3,000 BSA 3x0.16 = 0.48 3.84 48 H2O 3x4.84 = 14.52 116.16 1452 (Use the space in the last column to do your calculation dependent on sample number). 7) Give the MasterMix a thorough vortex and spin it down. 8) Spin down the plate with primers. 9) Add 15.0 µl to each well that will be used in the PCR. 10) Add 2 µl of the primer mix to each well. 11) Move to the other hood before adding 3 µl of the DNA template to each well with MasterMix. (If your samples are in the Genetic lab B310, you add the 3 µl of DNA in the Metabarcoding Workstation in B310). 12) Seal, wrap in bags, put in fridge while you clean the hoods following the cleaning protocol.