May 28, 2025

Public workspaceDNA extraction and DNA quality control from fish tissue using the QIAwave DNA Blood & Tissue Kit (QIAGEN) or NucleoSpin Tissue Kit (MACHEREY-NAGEL)

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmyoz9v3p/v1
DNA extraction and DNA quality control from fish tissue using the QIAwave DNA Blood & Tissue Kit (QIAGEN) or NucleoSpin Tissue Kit (MACHEREY-NAGEL)
  • Hervé Rogissart1,2,3,
  • Cecile Chardon1
  • 1UMR CARRTEL;
  • 2INRAE;
  • 3USMB
Hervé Rogissart
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvmyoz9v3p/v1
Protocol Citation: Hervé Rogissart, Cecile Chardon 2025. DNA extraction and DNA quality control from fish tissue using the QIAwave DNA Blood & Tissue Kit (QIAGEN) or NucleoSpin Tissue Kit (MACHEREY-NAGEL). protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmyoz9v3p/v1
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 20, 2025
Last Modified: May 28, 2025
Protocol Integer ID: 124736
Keywords: DNA extraction, Fish, Salmonids, dna quality control from fish tissue, dna extraction from other fish species, genomic dna from fish tissue, dna extraction procedure, summary sheet of the dna extraction procedure, dna extraction, qiawave dna blood, extracting genomic dna, fish tissue, dna quality control, dna quality, nucleospin tissue kit, other fish species, dna, fish, extraction, tissue kit, salvelinus alpinus
Abstract
This protocol describes a column-based method for extracting genomic DNA from fish tissue (e.g., Salvelinus alpinus) using either the QIAwave DNA Blood & Tissue Kit (QIAGEN) or the NucleoSpin Tissue kit (MACHEREY-NAGEL), adapted from the manufacturers’ recommendations. The protocol is also well-suited for DNA extraction from other fish species. A summary sheet of the DNA extraction procedure is included to support its practical implementation in the laboratory.
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Hervé Rogissart
Materials
  • Samples
- Fish tissue
- Preserved in ethanol (final concentration > 90 % of ethanol) or at Temperature-20 °C
- Weight needed = 2-10 mg

  • Reagents
To clean the workspace
- DNA/RNA-ExitusPlus
- Ethanol (70 %)

For prepare sample:
- DNA/RNA-ExitusPlus
- Ethanol 96 - 100 %
- Water

For DNA extraction
- DNA extraction kit: Qiagen Kit or Macherey-Nagel Kit
- Ethanol 96 - 100 %, molecular grade to prepare wash buffer
- Ultrapure water

  • Materials (excluding solutions preparation)
- Bunsen burner
- Chisel and lab pincer
- Balance
- Specific DNA-work station (sterile area equipped with air filtration)
- Microcentrifuge for 1.5 to 2 mL tubes (relative centrifugal force needed: 6000 to 19000 x g)
- Horizontal vortexer with microtube holder
- Vortexer
- Stove or dry bath (to have 56 °C)
- Pipettes: 100-1000 μL; 10-100 μL
- 2 trash cans: 1 for liquid and 1 for solid

  • Consumables
- Tips with filter:
> 1000 μL
> 100 μL
- 2 mL sterile microcentrifuge tube: 2 per sample
1 to prepare and lyse tissue (steps 1-2-3) and 1 to elute DNA (step 6)
- Gloves

Protocol materials
ReagentQubit™ dsDNA BR Assay KitThermo Fisher ScientificCatalog #Q32853
ReagentNucleoSpin TissueMacherey-NagelCatalog #740952.250
ReagentDNA/RNA-ExitusPlusApplychemCatalog #A7089
ReagentQIAwave DNA Blood & Tissue KitQiagenCatalog #69556
Troubleshooting
Safety warnings
Buffers used in these kits require safety precautions, mainly Buffer AL and Buffer AW1 or Buffer B3 and BW which contain guanidine salt.

Safety informations of all buffers are availabe at :
Qiawave DNA Blood & Tissue Kit (QIAGEN)
NucleoSpin Tissue (MACHEREY-NAGEL)
Before start
  • Wear gloves througout the extraction process
  1. Preheat dry bath or stove to Temperature56 °C
  2. Clean the workspace withReagentDNA/RNA-ExitusPlusApplychemCatalog #A7089 followed by Concentration70 % volume ethanol
  3. Unpack ReagentQIAwave DNA Blood & Tissue KitQiagenCatalog #69556 (Qiagen Kit)
or
ReagentNucleoSpin TissueMacherey-NagelCatalog #740952.250 (Macherey-Nagel Kit)
4. Check/Prepare buffers


5.1. If using Qiagen Kit
  • Buffer ATL and Buffer AL may precipitate during storage
- Warm to Temperature56 °C until fully dissolved
  • Before first use, prepare Buffer AW1, Buffer AW2 and Buffer AE as indicated on the manufacturer's protocol. The information below is for a 250 columns kit.

-> Buffer AW1: In a Amount250 mL sterile glass bottle, transfer the entire volume of Buffer AW1/C (Amount98 mL ) and add ethanol Concentration96-100 % volume (Amount130 mL ). Cap the glass bottle tightly and mix thoroughly by inverting the bottle several times. To label the glass bottle, use the enclosed label and transfer it onto the glass bottle.
The final volume of Buffer AW1 is Amount228 mL . This solution is stable for 1 year when stored closed at room temperature.

-> Buffer AW2: In a Amount250 mL sterile glass bottle, transfer the entire volume of Buffer AW2/C (Amount6 mL ) and add ultrapure water (Amount60 mL ) + ethanol Concentration96-100 % volume (Amount160 mL ). Cap the glass bottle tightly and mix thoroughly by inverting the bottle several times. To label the glass bottle, use the enclosed label and transfer it onto the glass bottle.
The final volume of Buffer AW2 is Amount226 mL . This solution is stable for 1 year when stored closed at room temperature.

-> Buffer AE: In a 150mL sterile glass bottle, transfer the entire volume of buffer AE/C Amount10 mL (10 mL) and add ultrapure water Amount110 mL . Cap the glass bottle tightly and mix thoroughly by inverting the bottle several times. To label the glass bottle, use the enclosed label and transfer it onto the glass bottle.
The final volume of Buffer AE is Amount120 mL .

  • Prepare aliquots of each solution as needed


5.2. If using Macherey-Nagel Kit
  • Buffer T1 and Buffer B3 may precipitate during storage
- Warm between Temperature50 °C and Temperature70 °C until fully dissolved
  • Before first use, prepare Buffer B5 and Proteinase K solution as indicated on the manufacturer's protocol.

-> Buffer B5: add appropriate volume of ethanol Concentration96-100 % volume to Buffer B5 Concentrate as indicated on the bottle and write the date on the bottle. This solution is stable for 1 year when stored closed at TemperatureRoom temperature .

-> Proteinase K solution: add appropriate volume of Proteinase Buffer PB (provided) to lyophilized Proteinase K. Ensure all powder is dissolved and solution is homogeneous. Transfert Proteinase K solution in 1.5 or 2 mL sterile microtube. This solution is stable for 6 months at Temperature-20 °C .

  • Prepare aliquots of each solution as needed

Prepare sample:
  • Before start
- For each sample, label a Amount2 mL sterile microcentrifuge tube (Eppendorf safe-lock) with an unique identifier
- Decontaminate cutting tools: clean with DNA ExitusPlus, rinse with water and cover with ethanol
- Sterilize cutting tools using bunsen burner
  • Cut up to approximatively 2-10 mg de tissue
  • Place tissue in annotated tube
Note
- Decontaminate or replace cutting tools for each sample to prevent cross-contamination
- Cutting the tissue into small pieces to enable more efficient lysis
- Store at Temperature+4 °C for quick use (< 8h) or at Temperature-20 °C for further use (> 8h)


Lyse sample 1/2:
  • Prepare the lysis mixture containing the following per sample
AB
Qiagen kitMacherey-Nagel kit
180 µL buffer ATL180 µL buffer T1
20 µL Proteinase K25 µL Proteinase K
Lysis mixture composition

Note
- Homogenize Buffer and Proteinase K
- When preparing the mix for multiple samples, allow a marge for 1-2 additional samples

  • Add lysis mixture to each sample
AB
Qiagen kitMacherey-Nagel kit
200 µL 205 µL
Lysis mixture volume / sample

  • Vortex briefly Duration00:00:05 by pulse-vortexing
  • Centrifuge Centrifigation, 00:00:05 in a microcentrifuge

Note
Check that tubes are tightly sealed to prevent evaporation during incubation, and ensure that tissue samples are fully immersed in the lysis buffer. If this was not the case, a brief centrifugation was performed to re-establish contact between the tissue and the solution.

  • Incubate at Temperature56 °C for at least Duration02:00:00 in a dry bath or in a stove

Note
- If using a thermomixer set toShaker300 rpm
- Make sure the incubator is preheated to 56°C otherwise the lysis time must be increasedDurationOvernight incubation is applied to ensure that digestion will be complete and/or to facilitate the organization of extraction.

Lyse sample 2/2 & Adujst DNA binding conditions:
  • To save time while waiting for the lysis incubation, per sample prepare and annotate:
- spin columns for DNA purification placed in waste tubes (provided)
- Amount2 mL microtubes for DNA elution

  • Few minutes before the end of incubation, prepare buffer and ethanol mixture containing the following per sample
AB
Qiagen kitMacherey-Nagel kit
200 µL AL200 µL B3
Buffer and ethanol mixture composition
Note
- Homogenize Buffer and ethanol mixture
- When preparing the mix for multiple samples, allow a marge for 1-2 additional samples
  • Each sample must be vortexed briefly Duration00:00:15 and centrifuged briefly Duration00:00:05
  • Add buffer and ethanol mixture
AB
Qiagen kitMacherey-Nagel kit
400 µL410 µL
Buffer and ethanol mixture volume / sample
  • Immediately vortex for Duration00:00:15 to mix and centrifuge Centrifigation, 00:00:05 in a microcentrifuge

Note
- It is important to immediately mix the samples by vortexing or pipetting after adding the Buffer and ethanol mixture to ensure a homogeneous solution. Failure to do so may affect DNA yield.

Bind DNA:
  • Transfer the mixture samples to spin column placed in a waste tube (provided and annotated)
  • Centrifuge at TemperatureRoom temperature to capture the DNA on the column membranes. Follow the parameters indicated in the table below.


ABC
Qiagen kitMacherey-Nagel kit
Column name DNeasy Mini Spin ColumnNucleoSpin Tissue Column
Centrifugation6000 x g - 1 min11000 x g - 1 min

  • Discard the flow-through and reuse the Waste Tube
Wash & Dry silica membrane:
  • To clean DNA, do 2 washes as described in the table below
  • Between each centrifugation, discard the flow-through and reuse the Waste Tube
  • To dry silica membrane, do an additional centrifugation described in table below
ABC
QiagenMN
1st wash500 µL AW1 6000 x g - 1 min500 µL BW 11000 x g - 1 min
2nd wash500 µL AW2 19000 x g - 2 min600 µL B5 11000 x g - 1 min
Dry silica membrane19000 x g - 1 min11000 x g - 1 min

Elute:
  • Place the Spin Column in a Amount2 mL annoted clean tube
- For each sample check the correspondence of Spin Column label with Amount2 mL tube label
  • Add Amount100 µL of elution buffer (provided in the kit) to the membrane of each column
AB
Qiagen kitMacherey-Nagel kit
Buffer AEBuffer BE
  • Incubate at TemperatureRoom temperature Duration00:01:00
  • To elute DNA from the membrane,centrifuge by respecting these instructions
AB
Qiagen kitMacherey-Nagel kit
6000 x g - 1 min11000 x g - 1 min
Safety information
- Position the lid inward the center of the centrifuge
- Caution do not discard the eluate
  • Eluting with Amount100 µL increases the DNA concentration but reduces the overall DNA yield


Pipetting
DNA storage:
DNA is ready to use immediately or store at:
  • Temperature4 °C for use a few days
  • Temperature-20 °C for use within a few weeks
  • Temperature-80 °C for long-term storage

Note
- Before using, allow the sample to equilibrate to TemperatureRoom temperature
- Perform pulse-vortexing for Duration00:00:05 followed by microcentrifugation

DNA quantification and DNA quality control:
NanoDrop measurement:

  • Use Amount2 µL of used elution buffer as blank as standard with the NanoDrop
  • To validate blank, add Amount2 µL to used elution buffer into the surface of the NanoDrop and read
Equipment
NanoDrop™ One UV-Vis Spectrophotometer
NAME
spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU
https://www.thermofisher.com/order/catalog/product/ND-ONE-W
LINK
Sample Volume (Metric): Minimum 1µL; Spectral Bandwidth: ≤1.8 nm (FWHM at Hg 254 nm); System Requirements: Windows™ 8.1 and 10, 64 bit; Voltage: 12 V (DC); Wavelength Range: 190–850 nm
SPECIFICATIONS
  • To measure DNA concentration of sample, add Amount2 µL of sample DNA into the surface of the NanoDrop and read
- Note down the A260/280, A260/230 and concentration (ng/μl)
- or export the data to a USB storage device
Note
- Clean the surface between each read


Pipetting
Wash
Analyze
Qubit Measurement:

  • Ensure the Qubit is calibrated before starting
Equipment
Invitrogen™ Qubit™ 3 Fluorometer
NAME
Accurately measures DNA, RNA, and protein using the highly sensitive fluorescence-based Qubit quantitation assays
TYPE
Invitrogen™ Q33216
BRAND
Q33216
SKU
https://www.fishersci.co.uk/shop/products/qubit-3-0-fluorometer/15387293
LINK
  • Use the ReagentQubit™ dsDNA BR Assay KitThermo Fisher ScientificCatalog #Q32853
- Analyze Amount1 µL of extracted DNA
- Note the concentration (ng/μl)