🧬

DNA Extraction - PowerSoil Pro

Introduction

The PowerSoil Pro Kit is used to extract high-quality DNA from microbial samples. This kit uses a variety of mechanisms (beads, heat, detergent) to efficiently break open microbial cells, which are typically resistant to lysis.

For GERA purposes, this protocol is only used in conjunction with Oxford Nanopore sequencing preps.

The Qiagen Handbook is here.

Safety

  • Follow universal lab safety precautions: wear a lab coat and gloves when working in the lab, no open-toed shoes, no eating and drinking.
  • DO NOT add bleach or acidic solutions to the buffers contained in the PowerSoil Pro Kit. Guanidine salts will react to create toxic gases.
  • Save liquid waste from Buffers CD1, CD2, and CD3 for disposal in Philadelphia.
  • Liquid waste from Buffers EA and CD5 can be disposed of in the regular garbage. Place in a closed container.
  • All components of the PowerSoil Pro Kit should be stored in a dry place at room temperature (15-30°C).

Reagents

Reagent
Vendor
Catalog
Storage
DNeasy PowerSoil Pro Kit
Qiagen
10223
Room Temp (except CD2 at 4°C)

Equipment and Consumables

Item
Vendor
Catalog
100 ml Analytical Filter Funnel w/ 0.2 um Filter
Thermo Scientific
1450020
1 L Filter Flask
Thermo Scientific
DS41011000
Rubber Stoppers for Flask
Fisher
14-135M
Hand Pump
Thermo Scientific
61320010
Homogenization Kit
Biomeme
3000093
BioShake IQ Thermo Mixer, with deep well plate adapter
QINSTRUMENTS
1808-0506
Mini Vortexer
Fisher
14-955-151
MySpin 12 mini centrifuge (max 12,500 rpm - 9,800 rcf)
Thermo Scientific
75004081
P1000, P200 Pipets and Filter Tips
NA
NA

Protocol

Water Sample Filtration

  1. Set up the filter apparatus with a new single-use analytical filter funnel containing a 0.2 um filter.
  2. Let the collected sample sit undisturbed for a few minutes to allow sand and debris to settle.
  3. Pour the water sample into the filter funnel to the 100 ml mark and use the vacuum pump to draw the water through the filter. Repeat with additional 100 ml volumes until the desired volume is reached. Record the total volume of water filtered.
💡
Note - Yield is ~1 ng DNA per ml water. 1-2 L is needed for Oxford Nanopore library prep.
  1. Remove the filter from the vacuum flask and discard the cup.
  2. Flip the filter and Whatman paper into your gloved hand or filter funnel top. Discard the Whatman paper and then roll up the filter with the transfer side facing inward.
  3. Transfer the rolled filter into a Biomeme homogenization tube.
  4. Add 1 ml of Solution CD1 to the homogenization tube containing the filter.
  5. Shake by hand for 1 minute.
  6. Transfer up to 800 ul to a PowerBead Pro tube.
  7. Vortex to mix.

Liquid Sample Pretreatment (Saliva, Sputum, Sewage, etc.)

  1. Add up to 200 ul liquid sample to a PowerBead Pro tube.
  2. Bring the total volume up to 800 ul with CD1.
  3. Vortex to mix.

Swab Sample Pretreatment (Fecal, Oral, Surface, etc)

  1. Using sterilized scissors, cut the swab tip into a PowerBead Pro tube.
  2. Add 800 ul of CD1 to the tube.
  3. Vortex to mix.

Extract Samples

💡
Save all the liquid waste and bring back to Philadelphia for proper disposal.
  1. Heat the PowerBead Pro tube containing sample and CD1 for 10 minutes at 65C.
  2. Vortex or hand shake for 10 minutes.
  3. Centrifuge the PowerBead Pro tube for 1.5 min at max speed in a MySpin 12 mini centrifuge (12,500 rpm).
  4. Transfer the supernatant to a new 2 ml tube. Expect 500-600 ul.
  5. Add 200 ul Solution CD2 and vortex.
  6. Centrifuge for 1.5 minutes at max speed in a MySpin 12 mini centrifuge (12,500 rpm).
  7. Avoiding the pellet, transfer up to 700 ul supernatant to a new 2 ml tube. Expect 500-600 ul.
  8. Add 600 ul of Solution CD3 and vortex for 5 seconds.
  9. Load 650 ul of lysate onto a MB Spin Column and centrifuge at max speed for 1 minute (or until solution goes through column).
  10. Discard the flow-through and repeat with the remaining lysate.
  11. Place the MB Spin Column into a new 2 ml collection tube.
  12. Add 500 ul Solution EA to the MB Spin Column and centrifuge at max speed for 1 minute (or until solution goes through column).
  13. Discard the flow-through and place the column into the same 2 ml collection tube.
  14. Add 500 ul Solution C5 to the MB Spin Column and centrifuge for 1 minute at max speed (or until solution goes through column).
  15. Discard the flow-through and place the column into a new 2 ml collection tube.
  16. Centrifuge for 3 minutes at max speed in a MySpin 12 mini centrifuge (12,500 rpm).
  17. Carefully place the MB Spin Column into a new 1.5 ml Elution Tube. Cut off the top of the 1.5 ml tube so that it doesn’t get destroyed in the centrifuge.
  18. Add 80 ul (50-100 ul) of Solution C6 to the center of the white filter membrane.
  19. Centrifuge for 3 minutes at max speed in a MySpin 12 mini centrifuge (12,500 rpm).
  20. (optional) Repeat steps 18 and 19 to increase yields.
  21. Eluted DNA can be used immediately or stored in the freezer.