May 28, 2024

Public workspaceDNA extraction NucleoSpin Tissue INRAE eWHALE

DOI
dx.doi.org/10.17504/protocols.io.rm7vzxexrgx1/v1
  • 1INRAE - DECOD
Teddy Urvois
Open access
DOI: dx.doi.org/10.17504/protocols.io.rm7vzxexrgx1/v1
Protocol Citation: Teddy Urvois, Anne-Laure Besnard, Erwan Quéméré 2024. DNA extraction NucleoSpin Tissue INRAE eWHALE . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxexrgx1/v1
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: January 03, 2024
Last Modified: May 28, 2024
Protocol Integer ID: 92898
Abstract
This DNA extraction protocol aims at extracting total genomic DNA from filtered seawater samples suspended in Tris-EDTA-SDS Buffer.

Our protocol mostly follows the NucleoSpin Tissue protocol (qr.mn-net.com/qr/(IFU)740952), section 5 (Standard protocol for human or animal tissue and cultured cells). It starts at step 3, with 200µL of sample, and adds 25µL Proteinase K to the original step. To maximize DNA concentration, Buffer BE is heated at 70°C, and elution is repeated twice with the same 100µL of Buffer BE with 3 min incubation.
Materials
- Pipettes : monochannel p100, p200, p1000 and corresponding filter tips
- 1.5 mL microcentrifuge tubes
- Ethanol (96-100%)
Before start
Sample collection and preparation:
Seawater was filtrated using Sylphium pods with 0.45µm filters. Using a syringe, water was removed and filters dried. To lyse cells and preserve DNA, 3mL of Tris-EDTA-SDS Buffer was added to each Sylphium pod. Sylphium pods were freezed quickly after.

Lab work:
Prepare Buffer B5 and Proteinase K.
Put all equipment (micropipettes, disposable tips, microtubes, columns, ethanol, B3, BW and B5) in UV cabinet and light UV for 20 mins.
Set incubator to 70°C and heat Buffer BE to 70 °C.
Vortex and spin samples.
Lyse sample
Lyse sample
Add Amount200 µL of SampleSample to a microcentrifuge tube.
Add Amount200 µL Buffer B3 and Amount25 µL Proteinase K solution.
Vortex vigorously.
Incubate at 70 °C for Duration00:10:00 .
Vortex briefly.
10m
Adjust DNA binding conditions
Adjust DNA binding conditions
Add Amount210 µL ethanol (96 – 100 %) to the sample and vortex vigorously.
Bind DNA
Bind DNA
For each SampleSample , place one NucleoSpin Tissue Column into a Collection Tube.
Apply the SampleSample to the column.
Centrifuge for Duration00:01:00 at 11,000 x g.
Discard Collection Tube with flowthrough and place the column in a new Collection Tube.
1m
Wash silica membrane
Wash silica membrane
Add Amount500 µL Buffer BW.
Centrifuge for Duration00:01:00 at 11,000 x g.
Discard flowthrough and place the column back into the Collection Tube.
Add Amount600 µL Buffer B5 to the column and centrifuge for Duration00:01:00 at 11,000 x g.
Discard flowthrough and place the column back into the Collection Tube.
2m
Elute highly pure DNA in two steps with Buffer BE heated at 70°C
Elute highly pure DNA in two steps with Buffer BE heated at 70°C
8m
Place the NucleoSpin Tissue Column into a 1.5 mL microcentrifuge tube and add Amount100 µL Buffer BE.
Incubate at room temperature for Duration00:03:00 .
Centrifuge Duration00:01:00 at 11,000 x g.
Add the same Amount100 µL Buffer BE back in the NucleoSpin Tissue Column.
Incubate at room temperature for Duration00:03:00 .
Centrifuge Duration00:01:00 at 11,000 x g.
Discard NucleoSpin Tissue Column.
8m