Soil DNA
Oxford Nanopore
Soil DNA
FOR RESEARCH USE ONLY
Contents
Introduction
Materials
Method
Results
Sequencing performance
Introduction
This protocol describes a method for DNA extraction from soil samples. We recommend extracting the DNA using the QIAGEN DNeasy PowerMax Soil Kit (formerly known as MO BIO PowerMax Soil DNA Isolation Kit) using 8 g of starting material.
We have tested modifications to the protocol, such as bead-beating at a high temperature (65°C) and overnight at room temperature (20 rpm). Neither of the methods presented an improvement to the yield.
The sequencing performance was assessed using the Rapid and Ligation Sequencing Kits, with and without SPRI size selection.
Materials
- Soil sample (~8 g)
- Qiagen DNeasy PowerMax Soil Kit/MO BIO PowerMax Soil DNA Isolation Kit
- Vortex mixer
- Centrifuge with capacity for 50 ml Falcon tubes
- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
Method
Start with 8 g of material. We do not recommend exceeding the kit capacity of 10 g, as it may result in membrane blockage.
Perform the lysis as recommend in the manufacturers’ manual, page 8. If you do not have a vortex adapter for Falcon tubes, tape the tubes to the vortex mixer for the bead-beating step. If using this method, ensure the tubes are not full, and that the content of each tube can be properly mixed.
Follow the Qiagen DNeasy PowerMax Soil Kit protocol.
We recommend eluting the DNA in 2 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to help preserve DNA integrity, and incubating for 20 minutes at room temperature before the final spin.
Perform a SPRI size selection with ~100 µl of eluate (corresponding to 3 µg of DNA).
Results
Before SPRI size selection:
Yield: 30-45 ng/µl (total yield 60-90 µg)
OD 260/280: 2.10
OD 260/230: 1.73
After SPRI size selection:Yield: 35-40 ng/µl (total yield 1.4-1.6 µg)
OD 260/280: 2.06
OD 260/230: 1.99
Fragment size (FEMTO pulse):Before SPRI size selection:
After SPRI size selection:
After SPRI size selection:
Fragment size (FEMTO pulse):
Sequencing performance
Libraries prepared using the Ligation Sequencing Kit.
- Read length profile with and without SPRI selection:
Libraries prepared using the Rapid Sequencing Kit
- Read length profile with and without SPRI selection:
