ITS2 amplicon PCR for Symbiodiniaceae (incl. touchdown options)

Amplify the Symbiodiniaceae ITS2 region from coral DNA for amplicon sequencing, with high-annealing and touchdown cycling options for low-symbiont (e.g. bleached) samples.

Version 1.0 · last updated 2026-10-05 · Used in 8 notebook entries ↓

At a glance

Input	Coral holobiont DNA diluted to 4 ng µL⁻¹
Output	ITS2 amplicons with Illumina overhangs, checked on a gel, ready for indexing/sequencing
Reaction	25 µL, Phusion master mix, in duplicate
Time	~2 h PCR + 1 h gel (estimate)
Tested on	Porites astreoides (healthy, partially dead and bleached); Montipora capitata

This page covers the PCR and gel check. Primer sequences, pooling and sequencing submission follow the Putnam Lab ITS2 sequencing protocol.

WarningLow-symbiont samples

When symbiont density is very low (e.g. bleached corals), the standard PCR can amplify coral DNA instead of ITS2: faint bands, or bands of the wrong size. The cycling options below raise annealing stringency to fix this.

Materials

• Phusion High-Fidelity PCR Master Mix (2×)
• ITS2 forward and reverse primers with Illumina adapter overhangs, 10 µM
• Ultrapure water; DNA at 4 ng µL⁻¹
• Positive control (DNA that amplified before) and a no-template negative control
• Thermocycler; agarose gel equipment (1% gel)

Procedure

A. Master mix

1. Number of reactions = (samples + 1 positive + 1 negative + 1 spare) × 2 (duplicate wells).

2. Per well:

   Reagent	µL
   Phusion Master Mix (2×)	12.5
   F primer (10 µM)	0.5
   R primer (10 µM)	0.5
   Ultrapure water	10.5
   Master mix total	24
   DNA (4 ng µL⁻¹)	1

   e.g. 4 samples + controls + spare = 7 × 2 = 14 wells → 175 µL Phusion, 7 µL each primer, 147 µL water.

3. Add 24 µL master mix per well, then 1 µL DNA (water for the negative). Seal, mix, and spin down.

B. Cycling: choose one

Program	Use when	Cycling
High-annealing	Primers with Illumina adapter tails (used Apr 2022)	95 °C 3 min → 35 × (95 °C 30 s, 71 °C 30 s, 72 °C 30 s) → 72 °C 2 min → 4 °C
TD-1 (Korbie & Mattick 2008, Tm 52 °C)	Low-symbiont samples	95 °C 3 min → 11 cycles of 95 °C 30 s / anneal 45 s stepping 62 → 52 °C (−1 °C per cycle) / 72 °C 60 s → 25 × at 52 °C → 72 °C 5 min
TD-2	Low-symbiont samples	As TD-1, but step down 62 → 47 °C (Tm − 5) over 16 cycles, then 20 × at 47 °C
TD-3 (modified from H. Putnam)	Low-symbiont samples	95 °C 7 min → 20 × (94 °C 30 s, 62 °C 30 s, −0.5 °C per cycle, 72 °C 1 min) → 12 × at 52 °C → 72 °C 7 min

The 71 °C annealing is the calculated Tm of the primers including the Illumina adapter tails, which is why it is more specific than the original program.

ImportantWhich program to use

In March 2022, TD-1 did not give clean bands; TD-2 and TD-3 were compared on a gel (2022-03-16). In April 2022, the high-annealing (71 °C) program was used for the Porites July Bleaching re-sequencing (2022-03-19). TODO: record which program gave the best bands and make it the default here.

C. Gel check

4. Run an aliquot of each reaction (e.g. 5 µL) on a 1% agarose gel (75–100 V) with a ladder.
5. Expect one clean band at the ITS2 amplicon size (TODO: add expected size with adapters) in samples and the positive control, and nothing in the negative.
6. Pool duplicate wells and continue with cleanup/indexing per the Putnam Lab protocol.

Troubleshooting

Problem	Likely cause	Fix
Faint or no band in bleached samples	Very few symbiont templates; coral DNA out-competes	Use a touchdown or 71 °C program; check DNA concentration
Band at the wrong size	Off-target (coral) amplification	Raise annealing stringency (71 °C or touchdown)
One sample much brighter than the rest	Uneven template	Re-check the 4 ng µL⁻¹ dilution
Band in the negative control	Contamination	New water and primer aliquots; clean the PCR area

References

• Korbie DJ & Mattick JS 2008. Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nature Protocols 3:1452–1456. Link
• Putnam Lab / E. Strand. ITS2 sequencing protocol
• Analysis: ITS2 central document

Notebook entries using this protocol

Changelog

Date	Version	Change
2026-10-05	1.0	Written up from the 2021–2022 ITS2 runs and touchdown tests
2022-04	–	71 °C annealing (Tm with adapters) for the Porites July Bleaching re-sequencing
2022-03	–	Touchdown programs TD-1 to TD-3 tested on low-symbiont samples
2021	–	Original Putnam Lab ITS2 PCR on Thermal Transplant and Porites July Bleaching samples