Protocol for 16S Amplicfication of coral and reef sediment with 515F and 806R V4 primers
The goal of running this procedure is to capture the V4 16S rDNA region of the microbial community in reef sediment for amplicon sequencing.
Materials and Methods
- Q5® Hot Start High-Fidelity 2X Master Mix (link)
- Invitrogen™ UltraPure™ DNase/RNase-Free Distilled Water (link)
- Forward and Reverse Amplification Primers (link)
- FWD: 515F (Parada et al., 2016) GTGYCAGCMGCCGCGGTAA
- REV: 809R (Apprill et al., 2015) GGACTACNVGGGTWTCTAAT
- Eppendof Mastercycler (catalog number 5333)
- Purified template DNA
- Pipetters and tips (p1000, p200, p10), multichannel is helpful but not necessary
- 0.2mL PCR Tube Strips
Samples
Take note of the following parameters in your lab notebook. Dilute all samples to 1 ng/µL, with a total quantity of 20µL. This PhysWeb calculator is a helpful tool for calculating dilutions. In many cases, the DNA will already be extracted for the samples. This spreadsheet has a list of all HI22 microbiome samples and their DNA conc. immediately after extraction.
Tests were run on:
| Extraction Date | Sample ID | Type | DNA Quantity (ng/µL) | Stock (µL) | Buffer (ultrapure H20; µL) |
|---|---|---|---|---|---|
| 20220618 | S5 | Reef Sediment | 1.57 | 12.74 | 7.26 |
| 20220618 | S23 | Reef Sediment | 11.55 | 1.73 | 18.27 |
| 20220618 | S11 | Reef Sediment | 6.09 | 3.29 | 16.71 |
| 20220618 | C61 | Mcap tissue | 49.2 | 0.41 | 19.59 |
| 20220618 | C76 | Mcap tissue | 18.9 | 1.06 | 18.94 |
| 20220618 | C33 | Mcap tissue | 51.0 | 0.39 | 19.61 |
| 20220618 | G93 | Gfas tissue | 25.0 | 0.80 | 19.20 |
| 20220618 | G94 | Gfas tissue | 23.7 | 0.84 | 19.16 |
| 20220618 | G95 | Gfas tissue | 17.0 | 1.18 | 18.82 |
| 20220618 | Blank | Ultrapure Water | 0 | N/A | N/A |
PCR Reaction Mix
To create enough solution for three aliquots of 7 samples, a blank, plus a little extra, I used the following recipe:
- 390 µL Q5 MasterMix
- 39 µL 10µM 515F Forward Primer
- 39 µL 10µM 806R Forward Primer
- 156 µL ultrapure water
The solution above was aliquoted into 0.2mL PCR Tube Strips (20µL each), then 5µL of template DNA was added to each tube.
Thermocycling parameters
| Step | Temp | Time |
| — | — | — |
| Initial denaturation | 98°C | 00:00:30 |
| Denaturation | 98°C | 00:00:15 |
| Annealing | 55°C | 00:01:00 |
| Extension | 72°C | 00:00:30 |
| Cycle | GOTO 2 | REP 35 |
| Final extension | 72°C | 00:10:00 |
| Hold | HOLD: 4°C | not specified |
Purification
Results
Gel
- Followed Bhattacharya Lab Agarose Gel protocol for a medium 1% agarose gel (link) with the Thermo Scientific GeneRuler 1 kb Plus DNA Ladder