📖 Project Overview

This repository documents the molecular identification of fire worms (Hermodice carunculata and Eurythoe complanata) using DNA extraction, PCR amplification, gel electrophoresis, and sequencing.
The goal is to:

• Establish reliable gDNA extraction protocols for polychaetes.
• Test Folmer and Geller COI primers for amplification efficiency.
• Sequence PCR products and confirm species identity via BLAST analysis.
• Provide reproducible lab notes and results for future reference.

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🧪 Sample Preparation

• Extraction date: 27.1.25
• Kit used: Quick DNA/RNA Miniprep Plus Kit (Zymo Research)
• Storage: Worms stored at -20 °C in EtOH
• Steps:
  • Washed in 1X PBS several times
  • Homogenized in 800 µl DNA/RNA shield
  • 300 µl extracted immediately, 500 µl stored at -20 °C
  • Added 0.25 mm glass beads, vortexed 1 min at 2000 rpm
  • Proteinase K digestion (30 µl buffer + 15 µl enzyme, 30 min RT)
  • Centrifuged, supernatant transferred, lysis and wash steps performed
  • DNA eluted in 100 µl DNase/RNase‑free water (55 °C incubation, 5 min)

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📊 NanoDrop Results

Sample	Date	Conc. (ng/µl)	260/280	260/230
J1	12/6/23	98.3	1.91	2.05
J18	28/5/23	32.3	1.98	1.64
J16	12/6/23	162.4	1.93	2.10
Small worms	7/10/24	128.7	1.96	2.28
J15	14/1/25	162.4	1.93	2.40
1‑02	14/1/25	95.0	1.97	1.96

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🧬 DNA Template Dilutions

Sample	Dilution	Final Conc. (ng/µl)
J1	1:5	19.66
J18	1:1.5	21.53
J16	1:8	20.3
Small worms	1:6	21.45
J15	1:8	20.3
1‑02	1:5	23.75
Control (Stylophora pistillata)	—	19.0
NTC	—	—

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🔬 Primers

• Folmer et al., 1994 (Tm: 55 °C)
  • LCO1490: 5'-ggtcaacaaatcataaagatattgg-3'
  • HC02198: 5'-taaacttcagggtgaccaaaaaatca-3'
• Degenerative Primers – Geller et al., 2013 (Tm: 54 °C)
  • jgLCO1490: 5'-TITCIACIAAYCAYAARGAYATTGG-3'
  • jgHCO2198: 5'-TAIACYTCIGGRTGICCRAARAAYCA-3'

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⚗️ PCR Setup

Reaction volume: 25 µl

Component	Volume (1X)	Final Conc.
GoTaq® Green Master Mix (2X)	12.5 µl	1X
Upstream primer (10 µM)	1.5 µl	0.6 µM
Downstream primer (10 µM)	1.5 µl	0.6 µM
H₂O	8.5 µl	—
DNA template (~20 ng)	1.0 µl	—

Cycler program (Applied Biosystems SimpliAmp):

• Initial denaturation: 94 °C, 2 min
• 34 cycles:
  • 94 °C, 45 s
  • 49 °C, 30 s
  • 72 °C, 1 min
• Final extension: 72 °C, 3 min

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🧫 Gel Electrophoresis

• Gel: 1% agarose in 0.5X TBE + RedSafe
• Run: 40 min at 110 V
• Ladders: 1 kb Perfect Plus, 100 bp Perfect DNA ladder
• Lane order:
  • Lanes 3–10: Folmer primers (J1, J18, J16, Small worms, J15, 1‑02, Stylophora, NTC)
  • Lanes 11–18: Degenerative primers (same sample set)
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📑 Sequencing Setup

Sample	Service	Primer	Size	Conc. (ng/µl)
Fireworm J1	PCR clean‑up + sequencing	djLCO1490	700	4
Fireworm J1	Sequencing	djHCO2198	700	4
Fireworm J18	PCR clean‑up + sequencing	djLCO1490	700	4
Fireworm J18	Sequencing	djHCO2198	700	4
Fireworm J16	PCR clean‑up + sequencing	djLCO1490	700	4
Fireworm J16	Sequencing	djHCO2198	700	4
Fireworm J15	PCR clean‑up + sequencing	djLCO1490	700	4
Fireworm J15	Sequencing	djHCO2198	700	4
Fireworm 1‑02	PCR clean‑up + sequencing	djLCO1490	700	4
Fireworm 1‑02	Sequencing	djHCO2198	700	4
Small fireworm	PCR clean‑up + sequencing	djLCO1490	700	4
Small fireworm	Sequencing	djHCO2198	700	4

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🧾 BLAST Results

Sample J15

• Top hit: Hermodice carunculata (marine fireworm) – 98.79% identity, 96% coverage
• Other matches: Sabella spallanzanii (97.71%), multiple Hermodice carunculata vouchers (99–100% identity)

Sample 1‑02

• Top hit: Eurythoe complanata – 98.49% identity, 96% coverage
• Other matches: Amphinomidae sp. (99.84%), Eurythoe cf. complanata isolates (98–99%)

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🔁 How to Reproduce

1. Sample Collection & Storage
   • Collect fire worms and store at -20 °C in ethanol.
2. DNA Extraction
   • Use Quick DNA/RNA Miniprep Plus Kit.
   • Homogenize tissue in DNA/RNA shield with glass beads.
   • Perform Proteinase K digestion, lysis, and wash steps.
   • Elute DNA in DNase/RNase‑free water.
3. Quality Check
   • Measure DNA concentration and purity using NanoDrop.
   • Dilute samples to ~20 ng/µl for PCR.
4. PCR Amplification
   • Prepare 25 µl reactions with GoTaq® Green Master Mix.
   • Use Folmer or Geller COI primers.
   • Run thermal cycling program 94 °C → 49 °C