TITLE: Sterivex DNA Extraction with PowerSoil Kit
AUTHORS: Molly A Moynihan

[DESCRIPTION]
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. 




[STEPS]
SECTION: DNA extraction of sterivex filter using PowerSoil kit
1. Thaw sterivex samples and remove any RNAlater or excess water from filter using a syringe.
SECTION: DNA extraction of sterivex filter using PowerSoil kit
2. 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. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
3. Using sterile razor blade, slice the filter into 6 lengthwise strips. Place strips into powersoil bead tube.
SECTION: DNA extraction of sterivex filter using PowerSoil kit
4. Add 60μL of solution C1 and vortex briefly. Incubate at 70°C and 500rpm for 10 minutes. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
5. Vortex at max speed. Re-incubate at 70◦C and 500rpm for 10 minutes. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
6. 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). 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
7. Centrifuge at 10,000g for 30 seconds. 

SECTION: DNA extraction of sterivex filter using PowerSoil kit
8. Transfer 800μL supernatant to a clean 2mL collection tube. 

SECTION: DNA extraction of sterivex filter using PowerSoil kit
9. Add 250μL solution C2, vortex for 5 seconds, incubation at 4°C for 5 minutes. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
10. Centrifuge at 10,000g for 1 minute at room temperature. 

SECTION: DNA extraction of sterivex filter using PowerSoil kit
11. Transfer 600μL supernatant to a clean 2mL collection tube. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
12. Add 200μL of solution C3, vortex briefly. Incubate at 4°C for 5 minutes. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
13. Centrifuge at 10,000g for 1 minute at room temperature. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
14. Transfer 750μL supernatant to a clean 2mL collection tube. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
15. Add 1200μL of solution C4 (shake to mix before use). Vortex for 5 seconds. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
16. 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. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
17. Add 500μL of solution C5, centrifuge at 10,000g for 30 seconds at room temperature. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
18. Discard flow through 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
19. Centrifuge again at 10,000g for 1 minute at room temperature. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
20. Carefully place spin filter in a clean 2ml collection tube. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
21. 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. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
22. Centrifuge at 10,000g for 30 seconds at room temperature. 
SECTION: DNA extraction of sterivex filter using PowerSoil kit
23. 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.