DNA extraction NucleoSpin Tissue INRAE eWHALE
Anne-Laure Besnard, Teddy Urvois, Erwan Quéméré
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.
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.
Steps
Lyse sample
Add 200µL of Sample to a microcentrifuge tube.
Add 200µL Buffer B3 and 25µL Proteinase K solution.
Vortex vigorously.
Incubate at 70 °C for 0h 10m 0s.
Vortex briefly.
Adjust DNA binding conditions
Add 210µL ethanol (96 – 100 %) to the sample and vortex vigorously.
Bind DNA
For each Sample, place one NucleoSpin Tissue Column into a Collection Tube.
Apply the Sample to the column.
Centrifuge for 0h 1m 0s at 11,000 x g.
Discard Collection Tube with flowthrough and place the column in a new Collection Tube.
Wash silica membrane
Add 500µL Buffer BW.
Centrifuge for 0h 1m 0s at 11,000 x g.
Discard flowthrough and place the column back into the Collection Tube.
Add 600µL Buffer B5 to the column and centrifuge for 0h 1m 0s at 11,000 x g.
Discard flowthrough and place the column back into the Collection Tube.
Elute highly pure DNA in two steps with Buffer BE heated at 70°C
Place the NucleoSpin Tissue Column into a 1.5 mL microcentrifuge tube and add 100µL Buffer BE.
Incubate at room temperature for 0h 3m 0s.
Centrifuge 0h 1m 0s at 11,000 x g.
Add the same 100µL Buffer BE back in the NucleoSpin Tissue Column.
Incubate at room temperature for 0h 3m 0s.
Centrifuge 0h 1m 0s at 11,000 x g.
Discard NucleoSpin Tissue Column.
