Aug 29, 2022
Sterivex DNA Extraction with PowerSoil Kit
This protocol is a draft, published without a DOI.
- 1Nanyang Technological University
Protocol Citation: Molly A Moynihan 2022. Sterivex DNA Extraction with PowerSoil Kit. protocols.io https://protocols.io/view/sterivex-dna-extraction-with-powersoil-kit-cfw4tpgw
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: August 29, 2022
Last Modified: August 29, 2022
Protocol Integer ID: 69308
Abstract
Adapted from: Jacobs, J., Rhodes, M., Sturgis, B. & Wood, B. 2009. Influence of environmental gradients on the abun- dance and distribution of Mycobacterium spp. in a coastal lagoon estuary. Appl. Environ. Microbiol., 73787384, DOI: 10.1128/AEM.01900-09
This protocol was used to extract DNA from sterivex filters collected during time series sampling in Singapore from 2017-2019 at Kusu and Hantu islands. The protocol follows instructions from the Qiagen Powersoil kit with the addition of phenol-chloroform-isoamyl alcohol between solutions C1 and C2.
Materials
Qiagen Power Soil DNA Exraction kit
Phenol-chloroform-isoamyl alcohol
Safety warnings
Use phenol-chloroform-isoamyl alcohol in fume hood
DNA extraction of sterivex filter using PowerSoil kit
DNA extraction of sterivex filter using PowerSoil kit
Thaw sterivex samples and remove any RNAlater or excess water from filter using a syringe.
Using sterile pliers, break open sterivex by applying pressure along the seam of the casing. Do so over a sterile space as the filter can pop out quickly.
Using sterile razor blade, slice the filter into 6 lengthwise strips. Place strips into powersoil bead tube.
Add 60μL of solution C1 and vortex briefly. Incubate at 70°C and 500rpm for 10 minutes.
Vortex at max speed. Re-incubate at 70◦C and 500rpm for 10 minutes.
In fumehood, add 700μL of room temperature phenol-chloroform-isoamyl alcohol to dissolve filter. Vortex at max speed for 10 minutes. (Steps 6, 8,9, 11, 12 should be done in a fumehood).
Centrifuge at 10,000g for 30 seconds.
Transfer 800μL supernatant to a clean 2mL collection tube.
Add 250μL solution C2, vortex for 5 seconds, incubation at 4°C for 5 minutes.
Centrifuge at 10,000g for 1 minute at room temperature.
Transfer 600μL supernatant to a clean 2mL collection tube.
Add 200μL of solution C3, vortex briefly. Incubate at 4°C for 5 minutes.
Centrifuge at 10,000g for 1 minute at room temperature.
Transfer 750μL supernatant to a clean 2mL collection tube.
Add 1200μL of solution C4 (shake to mix before use). Vortex for 5 seconds.
Load 675μL into spin filter, centrifuge at 10,000g for 1 minute at room temperature, discard flow through; add 675μL again to spin filter, centrifuge at 10,000g for 1 minute at room temperature, discard flow through. Load remaining supernatant into spin filter and centrifuge at 10,000g for 1 minute at room temperature, discard flow through.
Add 500μL of solution C5, centrifuge at 10,000g for 30 seconds at room temperature.
Discard flow through
Centrifuge again at 10,000g for 1 minute at room temperature.
Carefully place spin filter in a clean 2ml collection tube.
Add 60μL of nuclease free water to the center of the white filter membrane. (Adjust volume of nuclease free water based on expected yield and desired DNA concentration). Let sit for 5 minutes.
Centrifuge at 10,000g for 30 seconds at room temperature.
Discard spin filter. Aliquot DNA for Qubit and PCR. Store remaining sample at -20°C for short term storage or -80°C for long term storage.