Iva &Zcaron;u&zcaron;ul Vrgo&ccaron;; Ivana Lepen Plei&cacute;; Klara Ivaniševi&cacute;; Tanja Šegvić-Bubić

doi:10.21769/BioProtoc.5839

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Peer-reviewed

Optimized Phenol–Chloroform–Isoamyl DNA Extraction Protocol for Single Fish Eggs

IV Iva Žužul Vrgoč ^* email

IP Ivana Lepen Pleić ^*

KI Klara Ivanišević

TŠ Tanja Šegvić-Bubić

(*contributed equally to this work) 发布: 2026年10月20日第16卷第20期 DOI: 10.21769/BioProtoc.5839 浏览次数: 29

评审: Amr Galal Abdelraheem IbrahimAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of Estuarine Coastal and Shelf Science, featuring study using the protocol.

Aug 2026

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

Abstract

Reliable DNA extraction is essential for genetic research on marine species; however, obtaining sufficient DNA from single fish eggs remains challenging. Existing protocols often require optimization to achieve high PCR efficiency. The optimized phenol–chloroform–isoamyl extraction protocol presented in this paper improves DNA yield and quality from individual eggs of Atlantic bluefin tuna (Thunnus thynnus), bogue (Boops bops), saddled seabream (Oblada melanura), and painted comber (Serranus scriba) by modifying buffer volumes, incubation times, and washing steps, following prior micropuncturing of eggs on a glass slide. DNA quality is confirmed by spectrophotometry, PCR amplification of the mitochondrial COI gene, electrophoresis, and Sanger sequencing. This method provides a low-cost and effective approach for species identification from individual fish eggs.

Key features

• Effective and low-cost DNA extraction method from fish eggs.

• Uses only standard reagents and equipment, allowing wide application in all laboratories.

• High PCR efficiency.

Keywords: Atlantic bluefin tuna (Thunnus thynnus)

Fish eggs

Phenol–chloroform DNA extraction

Mitochondrial COI gene

Species identification

Graphical overview

Optimized phenol–chloroform–isoamyl protocol for DNA extraction from single fish eggs. Includes egg visualization under a stereomicroscope, micropuncture on a glass slide, transfer to lysis buffer, Proteinase K digestion, phenol–chloroform extraction, ethanol washing, DNA resuspension and storage at -20 °C, PCR amplification, and Sanger sequencing.

Background

Today, almost all methods for stock research on marine species rely on genetic studies. To obtain high-quality DNA, effective DNA extraction is required. There are many reliable protocols and commercial kits for isolating DNA from tissue, which provide high yields and excellent quality DNA. However, DNA extraction from a single fish egg may result in lower yields due to uniform cell stages. Aranishi (2006) [1] used a one-tube method for DNA extraction, while Golotin et al. (2023) [2] described a low-cost, non-enzymatic protocol for isolating embryonic cells from fish eggs. In our samples, the existing protocols did not consistently provide sufficient DNA yield for reliable PCR amplification from individual fish eggs. Therefore, further optimization was required to achieve reliable PCR amplification and to develop an optimized protocol for DNA extraction from individual fish eggs collected in the vicinity of Atlantic bluefin tuna (Thunnus thynnus) aquaculture farms. The primary aim of the sampling was to confirm the occurrence of T. thynnus spawning within the aquaculture cages. However, the area surrounding aquaculture facilities is also inhabited by a diverse assemblage of wild fish species, as aquaculture cages can attract wild fish by providing increased food availability and serving as functional marine protected areas [3]. Consequently, eggs of other teleost fish species were also collected during sampling conducted around the cages. These eggs were initially selected under a stereomicroscope based on their morphological characteristics and size, while their species identity was subsequently confirmed by molecular analysis. Because eggs of different fish species may occur simultaneously in the vicinity of aquaculture cages, and morphological characteristics alone may not be sufficient for reliable species identification, an efficient DNA extraction protocol for individual fish eggs is required. The protocol described here incorporates micropuncturing of eggs on a glass slide (see General note 1), reducing the volume of lysis buffer to 200 μL, adjusting the volume of phenol–chloroform–isoamyl alcohol, optimizing the incubation period (2 h), and extending the washing period to overnight.

Materials and reagents

Biological materials

1. Fish eggs

Reagents

1. 96% alcohol (Sigma-Aldrich, catalog number: 32294), storage: 4 °C

2. Phenol–chloroform–isoamyl alcohol mixture (Sigma-Aldrich, catalog number: 77617), storage: 4 °C

3. Proteinase K, 20 mg/mL (Thermo Fisher, catalog number: AM2542), storage: -20 °C

4. TE buffer (Thermo Fisher, catalog number: 12090015), storage: 4 °C

5. HotStartTaq DNA Polymerase kit (Qiagen, catalog number: 203203), storage: -20 °C

6. SYBR Safe (Thermo Fisher, catalog number: S33102), storage: room temperature

7. Agarose (Sigma-Aldrich, catalog number: 9012-36-6), storage: room temperature

8. DNA ladder (Cleaver Scientific Ltd., catalog number: CSL-MDNA-100BP), storage: 4 °C

9. GelPilot DNA loading dye, 5× (Qiagen, catalog number: 239901), storage: 4 °C

10. Milli-Q water (MQH₂O), storage: room temperature

11. 1 M Tris-HCl, pH 8.0 (Thermo Fisher, catalog number: J22638.AP), storage: room temperature

12. 0.5 M EDTA, pH 8.0 (Thermo Fisher, catalog number: R1021), storage: room temperature

13. 5 M NaCl, RNase-free (Invitrogen, catalog number: AM9760G), storage: room temperature

14. 10% SDS (Invitrogen, catalog number: 15553027), storage: room temperature

Solutions

1. Lysis buffer (see Recipes)

2. 70% alcohol (see Recipes)

3. Proteinase K, 20 mg/mL (see Recipes)

Recipes

1. Lysis buffer

Reagent	Final concentration	Quantity or volume
1 M Tris-HCl	10 mM	2 mL
0.5 M EDTA	1.25 mM	0.5 mL
5 M NaCl	150 mM	6 mL
10% SDS	0.2%	4 mL
H₂O	n/a	184 mL
Total	n/a	200 mL

Store the prepared lysis buffer at room temperature and use within 12 months.

2. 70% alcohol

Reagent	Final concentration	Quantity or volume
Ethanol (96%)	70%	36.5 mL
Distilled H₂O		to a final volume of 50 mL
Total	n/a	50 mL

Store the prepared 70% alcohol at 4 °C.

3. Proteinase K, 20 mg/mL

Reagent	Final concentration	Quantity or volume
Proteinase K powder	20 mg/mL	100 mg
MQH₂O		5 mL
Total	n/a	5 mL

Add MQH₂O to the powder and mix gently by inversion or pipetting. Avoid vigorous vortexing to prevent shearing the enzyme. Divide the 5-mL solution into small working aliquots (e.g., from 100 μL to 1 mL) to avoid repeated freeze/thaw cycles. Store liquid aliquots at -20 °C.

Laboratory supplies

1. Safe-lock 1.5 mL tubes (Eppendorf, catalog number: 0030120086)

2. PCR tubes (Eppendorf, catalog number: 0030124332)

3. Falcon 50 mL (Eppendorf, catalog number: 0030122178)

4. Pipette tips, 1–20 µL, 20–200 µL, 200–1,000 µL (Gilson, catalog numbers: F171203, F171503, F171703)

5. Laboratory gloves

6. Laboratory scissors

7. Histology glass slides (Thermo Scientific, catalog number: 960004)

8. Medical sterile needles: 22G × 1+14″ (0.7 × 30 mm) (black) and 21G × 1 + 12″ (0.8 × 40 mm) (green) (Sterican^®, B. Braun, Germany; catalog numbers: 4657624 and 4657527)

Note: Both 21G and 22G needles are suitable for egg micropuncture; therefore, no specific gauge recommendation based on egg size is required.

Equipment

1. Pipettes (Gilson, model: Pipetman L, FA10002M; FA10005M)

2. UV air recirculator (BioSan, model: UVR-Mi)

3. Stereomicroscope (Olympus, model: SZX12) with DeltaPix camera (model: HDMI16AMDPX)

4. Laboratory rack

5. Isolation and PCR cabinet (BioSan, model: UVT-S-AR)

6. Thermoshaker (BioSan, model: TS-100c)

7. Vortex (BioSan, model: V-1 plus)

8. Centrifuge (Eppendorf, model: 5430 R)

9. Mini centrifuge (Boeco, model: M-6)

10. Spectrophotometer (IMPLEN, model: N50)

11. PCR (Eppendorf, model: nexus GX2)

12. Electrophoresis system (Cleaver Scientific, model: microDOC-CSL-MDOCUV254)

Software and datasets

1. BioEdit-Biological sequence alignment editor, version 5.0.9 (Hall, 1999) [4]

2. BLAST (Basic Local Alignment Search Tool) (National Center for Biotechnology Information; free to use); https://blast.ncbi.nlm.nih.gov/Blast.cgi

Procedure

文章信息

稿件历史记录

提交日期: May 30, 2026

接收日期: Sep 7, 2026

在线发布日期: Sep 15, 2026

出版日期: Oct 20, 2026

版权信息

如何引用

Žužul Vrgoč, I., Lepen Pleić, I., Ivanišević, K. and Šegvić-Bubić, T. (2026). Optimized Phenol–Chloroform–Isoamyl DNA Extraction Protocol for Single Fish Eggs. Bio-protocol 16(20): e5839. DOI: 10.21769/BioProtoc.5839.