TITLE: PCR amplification of 16S rRNA, 18S rRNA, and nifH genes in coral samples
AUTHORS: Molly Moynihan

[STEPS]
?. [Primers]
ABCD1GenePrimerSequenceReference216S rRNAB969F 5'-ACGCGHNRAACCTTACC-3'Comeau 2011316S rRNABA1406R5'-ACGGGCRGTGWGTRCAA-3'Comeau 20114518S rRNAUNonMetF5'-GTGCCAGCAGCCGCG-3'Bower 2004618S rRNAUNonMetR5'-TTTAAGTTTCAGCCTTGCG-3'Bower 20047818S rRNAV4 18S For5'-CCAGCASCYGCGGTAATTCC-3'Piredda 2017918S rRNAV4 18S Rev5'-ACTTTCGTTCTTGATYRATGA-3'Piredda 20171011nifHIGK3-F5'-GCIWTHTAYGGIAARGGIGGIATHGGIAA-3'Gaby & Buckley 201212nifHDVV-R5'-ATIGCRAAICCICCRCAIACIACRTC-3'Gaby & Buckley 2012
ABCD1GenePrimerSequenceReference216S rRNAB969F 5'-ACGCGHNRAACCTTACC-3'Comeau 2011316S rRNABA1406R5'-ACGGGCRGTGWGTRCAA-3'Comeau 20114518S rRNAUNonMetF5'-GTGCCAGCAGCCGCG-3'Bower 2004618S rRNAUNonMetR5'-TTTAAGTTTCAGCCTTGCG-3'Bower 20047818S rRNAV4 18S For5'-CCAGCASCYGCGGTAATTCC-3'Piredda 2017918S rRNAV4 18S Rev5'-ACTTTCGTTCTTGATYRATGA-3'Piredda 20171011nifHIGK3-F5'-GCIWTHTAYGGIAARGGIGGIATHGGIAA-3'Gaby & Buckley 201212nifHDVV-R5'-ATIGCRAAICCICCRCAIACIACRTC-3'Gaby & Buckley 2012
?. [Set up and tips]
Clean all surfaces and pipettes with 10% bleach and 70% ethanol. Use a separate set of pipettes for preparing the master mix and for loading the template. Ideally, one set stays in UV hood and the other set on the bench.
?. [Set up and tips]
Place PCR clean microcentrifuge tubes (for making master mix), PCR plate or tubes,  and PCR lids or seals in UV hood, along with pipettes and pipette tips to be used in preparing the master mix. Turn UV light on for 15 minutes.
?. [Set up and tips]
While UV hood light is on, take out reagents from freezer and thaw. Mix and spin down after thawed.
?. [Set up and tips]
Use filter pipette tips to avoid contamination of your reagents and contamination between samples.
?. [16S]
In UV hood, prepare the following master mix in a PCR clean microcentrifuge tube. For each sample, perform PCR in duplicate reactions (20µl volume). Include at least 1x negative control and 1x positive control. Always prepare several extra reactions, as some solution will be lost during pipetting. If using 96-well plate and multichannel pipette reservoir, prepare at least 5 extra reactions.For N samples, multiply the following volumes by X, where X = 2xN + (# negative controls) + (# positive controls) + (# spare reactions) (0.4mM final concentration) (0.4mM final concentration)  (2mM final concentration)  (5% v/v final concentration)+ DNA template (10-15ng in 20µl reaction)+ nuclease free water to a final volume of 20µl
[HotStarTaq Master Mix ]
[10mM B696F]
[10mM BA1406R]
[25mM MgCl2]
[DMSO]
?. [Set up and tips]
If primers have not been diluted, aliquot and dilute to 10µM, in clean & sterilized UV hood.For primers at 100µM, add 10µl of primer to 90µl of nuclease free water.
?. [Set up and tips]
Only open PCR reagents (Polymerase, BSA, MgCl2, DMSO, nuclease free water, primers, etc.) in clean UV hood. Always use filter tips when working with these reagents. If possible, work with aliquots.
?. [Set up and tips]
NEVER bring DNA template into UV hood used to make master mix.
?. [16S]
Dispense master mix from Step 9 into PCR wells inside UV hood. Add nuclease free water to negative controls inside UV hood. Outside of UV hood, add DNA template and positive controls to PCR wells. Spin down PCR plate/tubes after all wells have been loaded and sealed.
?. [16S]
Perform PCR in thermocycler using the following profile:   ABCD1Temperature (°C)TimeCycles2955 min1xInitial Denaturation3941 min30xDenaturation4521 min30xPrimer Annealing5721 min30xExtension67210 min1xFinal Extension74∞1xHold
ABCD1Temperature (°C)TimeCycles2955 min1xInitial Denaturation3941 min30xDenaturation4521 min30xPrimer Annealing5721 min30xExtension67210 min1xFinal Extension74∞1xHold
?. [16S]
Pool duplicate samples. Perform gel electrophoresis to check results of PCR. Clean PCR products (if required by sequencing facility). See clean-up protocol below.
?. [UNonMet & 18S]
For coral (or other metazoan) samples, perform nested PCR with UNonMet primers followed by V4 18S primers. A clean-up step is necessary between the two PCR reactions.
?. [UNonMet & 18S]
In UV hood, prepare the following master mix in a PCR clean microcentrifuge tube. For each sample, perform PCR in duplicate 10µl reactions. Include at least 1x negative control and 1x positive control. Always prepare several extra reactions, as some solution will be lost during pipetting. If using 96-well plate and multichannel pipette reservoir, prepare at least 5 extra reactions.For N samples, multiply the following volumes by X, where X = 2xN + (# negative controls) + (# positive controls) + (# spare reactions) (0.4mM final concentration) (0.4mM final concentration)+ DNA template (5-10ng)+ nuclease free water to a final volume of 10µl
[HotStarTaq Master Mix ]
[10mM UNonMet F]
[10mM UNonMet R]
?. [UNonMet & 18S]
Dispense master mix from Step 14 into PCR wells inside UV hood. Add nuclease free water to negative controls inside UV hood. Outside of UV hood, add DNA template and positive controls to PCR wells. Spin down PCR plate/tubes after all wells have been loaded and sealed.
?. [UNonMet & 18S]
Perform PCR in thermocycler using the following profile:   ABCD1Temperature (°C)TimeCycles2955 min1xInitial Denaturation3941 min35xDenaturation451.11 min35xPrimer Annealing5721 min35xExtension67210 min1xFinal Extension74∞1xHold
ABCD1Temperature (°C)TimeCycles2955 min1xInitial Denaturation3941 min35xDenaturation451.11 min35xPrimer Annealing5721 min35xExtension67210 min1xFinal Extension74∞1xHold
?. [UNonMet & 18S]
Clean UNonMet PCR products with AMPure XP beads to remove primers. If the sample is not cleaned, primers from the first round will continue to amplify in the second PCR and there will be two bands (~600bp & ~400 bp). See AMPure XP beads clean-up below.
?. [UNonMet & 18S]
In UV hood, prepare the following master mix in a PCR clean microcentrifuge tube. Include at least 1x 18S negative control, 1x 18S positive control, 1x negative control from UNonMet PCR, and 1x positive control from UNonMet PCR. (Do not pool duplicates from UNonMet PCR, keep them as duplicates for 18S PCR).For N reactions from the UNonMet PCR, multiply the following volumes by X, where X = N + (# 18S negative controls) + (# 18S positive controls) + (# spare reactions) (0.4mM final concentration) (0.4mM final concentration)2ul of cleaned UNonMet PCR product+ nuclease free water to a final volume of 20µl
[HotStarTaq Master Mix ]
[10mM V4 18S For]
[10mM V4 18S Rev]
?. [UNonMet & 18S]
Dispense master mix from Step 18 into PCR wells inside UV hood. Add nuclease free water to 18S negative controls inside UV hood. Outside of UV hood, add cleaned PCR products from UNonMet PCR (including UNonMet positive & negative controls), and 18S  positive controls. Spin down PCR plate/tubes after all wells have been loaded and sealed.
?. [UNonMet & 18S]
Perform PCR in thermocycler using the following profile:   ABCD1Temperature (°C)TimeCycles2955 min1xInitial Denaturation3941 min25xDenaturation4521 min25xPrimer Annealing5721 min25xExtension67210 min1xFinal Extension74∞1xHold
ABCD1Temperature (°C)TimeCycles2955 min1xInitial Denaturation3941 min25xDenaturation4521 min25xPrimer Annealing5721 min25xExtension67210 min1xFinal Extension74∞1xHold
?. [UNonMet & 18S]
Pool duplicate samples. Perform gel electrophoresis to check results of PCR. Clean PCR products (if required by sequencing facility). See clean-up protocol below.
?. [nifH]
In UV hood, prepare the following master mix in a PCR clean microcentrifuge tube. For each sample, perform PCR in duplicate 20µl reactions. Include at least 1x negative control and 1x positive control. Always prepare several extra reactions, as some solution will be lost during pipetting. If using 96-well plate and multichannel pipette reservoir, prepare at least 5 extra reactions.For N samples, multiply the following volumes by X, where X = 2xN + (# negative controls) + (# positive controls) + (# spare reactions) (0.4mM final concentration) (0.4mM final concentration)  (4mM final concentration)+ DNA template (aim for ~20ng in 20µl reaction)+ nuclease free water to a final volume of 20µlNote: Higher template concentration improved amplification of the nifH gene in coral samples. If working with seawater, sediment, etc. template concentration may need to be reduced.
[HotStarTaq Master Mix ]
[10mM IGK3]
[10mM DVV]
[25mM MgCl2]
[BSA (2ug/ul)]
?. [nifH]
Dispense master mix from Step 22 into PCR wells inside UV hood. Add nuclease free water to negative controls inside UV hood. Outside of UV hood, add DNA template and positive controls to PCR wells. Spin down PCR plate/tubes after all wells have been loaded and sealed.
?. [nifH]
Perform PCR in thermocycler using the following profile:   ABCD1Temperature (°C)TimeCycles2955 min1xInitial Denaturation3941 min35xDenaturation4551 min35xPrimer Annealing5721 min35xExtension67210 min1xFinal Extension74∞1xHold
ABCD1Temperature (°C)TimeCycles2955 min1xInitial Denaturation3941 min35xDenaturation4551 min35xPrimer Annealing5721 min35xExtension67210 min1xFinal Extension74∞1xHold
?. [nifH]
Pool duplicate samples. Perform gel electrophoresis to check results of PCR. Clean PCR products (if required by sequencing facility). See clean-up protocol below. Note: IGK3/DVV primers + the overhang adapters may form dimers. This was observed when using one specific overhang adapter sequence, but not in another. Clean-up with 0.8-0.9x Ampure XP beads can remove these adapter/primer dimers.
?. [AMPure XP Bead Clean Up]
Bring AMPure XP beads to room temperature.
?. [AMPure XP Bead Clean Up]
Vortex beads for  before use. Add AMPure XP beads in desired bead/sample ratio. When adding beads to sample, mix by gently pipetting up and down 10x.Recommended for nested UNonMet clean-up after 1st PCR: 1.2x bead/sample.Recommended for nifH IGK3/DVV: 0.9x bead/sample.
?. [AMPure XP Bead Clean Up]
Incubate for  at .
0 Room temperature
?. [AMPure XP Bead Clean Up]
Place samples on magnetic stand and incubate for  at  or until the supernatant is clear.
0 Room temperature
?. [AMPure XP Bead Clean Up]
Leaving samples on the magnetic stand, carefully remove and discard the supernatant without disturbing the beads. Leave some of supernatant behind to avoid disturbing sample.
?. [AMPure XP Bead Clean Up]
Leaving samples on the magnetic stand, wash beads 2x with ethanol:
?. [AMPure XP Bead Clean Up]
Add  to each sample.
[70% Ethanol]
?. [AMPure XP Bead Clean Up]
Incubate on magnetic stand for .
?. [AMPure XP Bead Clean Up]
Remove and discard the supernatant without disturbing beads. Repeat
?. [AMPure XP Bead Clean Up]
After second wash, use a P20 or P10 pipette to completely remove any remaining ethanol.
?. [AMPure XP Bead Clean Up]
Prepare  using molecular biology grade ethanol. (Each sample will need  of 70% ethanol).
[ethanol]
400 µl
?. [AMPure XP Bead Clean Up]
Keep samples on the magnetic stand and allow beads to air dry for 5-10 minutes. The beads will go from shiny to matte. Do not over dry. If left for too long, the beads will appear to crack.
?. [AMPure XP Bead Clean Up]
Remove samples from the magnetic stand and add  to each sample. Resuspend beads by gently pipetting up and down 10x.Note: Adding less than 40μl will require extra mixing to insure liquid comes into contact with beads.
[nuclease free water]
?. [AMPure XP Bead Clean Up]
Incubate at  for .
0 Room temperature
?. [AMPure XP Bead Clean Up]
Place the samples on the magnetic stand for  or until the supernatant has completely cleared.
?. [AMPure XP Bead Clean Up]
Carefully transfer  of the supernatant to a new tube/plate, taking care not to transfer any of the beads.
40 µl