TITLE: DNA Extraction from 0.22µm Sterivex Filters - Qiagen Blood and Tissue with Inhibitor Removal
AUTHORS: Andreas Novotny

[DESCRIPTION]
This protocol is used to extract genomic DNA from 0.22μm sterivex filters using Quiagens Blood and Tissue Kit. The protocol was developed as an equivalent replacement for the Phenol Chloroform extraction protocol. An additional PCR inhibitor removal step has been added to increase reproducibility for PCR-based downstream methods. This is especially important for coastal waters influenced by land runoff of glacial debris. This protocol is developed to work across all domains of life, from viruses to prokaryotes to eukaryotes, allowing for both amplicon sequencing and shotgun sequencing.

[BEFORE_START]
Read background information, MIOP and BePOP-OBON information under the "Guidelines" tab.

[GUIDELINES]
 MIOP: Minimum Information about an Omics Protocol
 
 
 
 
AUTHORS
 

RELATED PROTOCOLS
 
 
ACRONYMS AND ABBREVIATIONS
 
 
GLOSSARY
 
 

BACKGROUND

This protocol is used to extract genomic DNA from 0.22μl sterivex filters using a modified version of the protocol developed by the Hakai Institute:
 
This protocol was developed as an equivavelt to the Phenol Chloroform protocol for newly started projects that does not require comparable results to the weekly sampling series. The protocol was also modified to take extra precaution to PCR inhibitors originating from turbid coastal waters. We can see that adding PCR inhibition removal substantiallyincreases PCR efficiencies of all samples, but especially samples from turbid waters. 

Spatial coverage and environments of relevance
This protocol was implemented as part of the Urban Ocean Bioidiversity project, a collaboration between University of British COlumbias Institute for the Oceans and Fistheries, and Tsleil-Waututh First Nation. In 2023 we sambled multiple stations in the Burrard Inlet BC, Canada (from Point Grey to Port Moody Arm, Including the Port of Vancouver). Samples were collected at four seasons. This protocol yielded DNA with successful 12S amplification rates for over 95% of the samples, including those from the glacial freshed in late spring.

Personnel Required
 1 Technician

Safety
Identify hazards associated with the procedure and specify protective equipment and safety training required to safely execute the procedure!

Training requirements
Sterile technique, pipetting skills. Work-safe laboratory practices.

Time needed to execute the procedure
1 Day (for 24 samples).

[STEPS]
SECTION: PREPARATION
1. 
This protocol assumes that samples have been collected and preserved according to this procedure:
 

SECTION: PREPARATION
5. Set aside ATL buffer, AL buffer, proteinase K, RNase A and anhydrous ethanol. 
Put the ATL and AL buffers in the incubator to eliminate any precipitate that may be in the solution. 
There is a cardboard box in the mini freezer with pro K aliquots. Each aliquot has enough volume for 12 samples. The anhydrous ethanol is stored in the CR1 mini fridge, and in the CR2 in the mini freezer.
SECTION: PREPARATION
6. Calculate the volume of each reagent you will need for each step and have a one-time-use tube to make an aliquot for that specific reagent. Try to add a little bit more than you need and dispose of the leftover. Prepare the aliquots inside the workstation (with HEPA filter).
SECTION: PREPARATION
7. Cut the parafilm to a size of 1 cm x 5 cm, two or three per Sterivex – sometimes you will need to replace the parafilm in the Sterivex.
SECTION: PREPARATION
4. Wipe down the benches, centrifuge, and working areas using the PREempt solution/wipes. 
Turn on the incubator and set the temperature to 56°C.
SECTION: BEADS BEATING AND INITIAL INCUBATION
8. Thaw the Sterivex filters.
SECTION: BEADS BEATING AND INITIAL INCUBATION
9. Remove the parafilm and remove Longmire’s buffer/SLB using a syringe. You can use a plastic “tripour” to dispose the buffer and then pour all of the volume in the sink. Rinse the tripour with water after use, dry and wipe it down using a paper towel and PREempt solution.
SECTION: BEADS BEATING AND INITIAL INCUBATION
10. If needed, dry the Sterivex inlet using a Kimwipes (one wipe per Sterivex). Place the Sterivex on a clean Kimwipe while preparing the other one Sterivexes.
SECTION: BEADS BEATING AND INITIAL INCUBATION
11. Add 0.1 mm silica beads (about 0.3 g or three spoons*) into the Sterivex using a weighting paper (make a funnel with the paper to slide in the beads, use one per Sterivex). *It is a white spoon that is stored in the drawer with the silica beads.
SECTION: BEADS BEATING AND INITIAL INCUBATION
12. Seal the outlet port of the Sterivex filter unit with the parafilm.
SECTION: BEADS BEATING AND INITIAL INCUBATION
13. Inject 720 μL ATL buffer into the Sterivex.

SECTION: BEADS BEATING AND INITIAL INCUBATION
14. One more Sterivex +ATL buffer should be prepared for the extraction blank for detecting contamination during DNA extraction.
SECTION: BEADS BEATING AND INITIAL INCUBATION
15. Place the Sterivex in the vortex adapter and do the “bead-beating” for 10 min to promote cell lysis.
SECTION: BEADS BEATING AND INITIAL INCUBATION
16. Get one 50mL tube per sample, and put the Sterivexes inside the 50mL tube. 
Incubate the Sterivex at 56°C for 30 min while tilting or rotating.
SECTION: BEADS BEATING AND INITIAL INCUBATION
17. Repeat steps 15 and 16 one more time.
  

SECTION: DIGESTION WITH PRO-K AND INCUBATION
18. Add  80 μL proteinase-K to each Sterivex.

SECTION: DIGESTION WITH PRO-K AND INCUBATION
19. Seal the outlet port of the Sterivex filter unit with a parafilm if you notice that some lysate is leaking.
SECTION: DIGESTION WITH PRO-K AND INCUBATION
20. Vortex tubes for ~10 s (or simply move it to mix the solution). 
Place the Sterivexes back to the 50mL.
Incubate tubes at 56°C for 2h while rotating.

SECTION: COLLECTING THE LYSATE
21. While warming the Sterivex filter unit to 56°C, prepare a 2.0 mL tube for DNA recovery (loBind DNA tubes). Note: write the necessary information on the cap of the 2.0 mL tube.

SECTION: COLLECTING THE LYSATE
22. After completion of warming, carefully remove the parafilm and the luer fitting on the inlet port of the Sterivex filter unit, while preventing liquid inside from leaking.
SECTION: COLLECTING THE LYSATE
23. Insert the inlet port of the Sterivex filter unit into the 2.0 mL tube and lightly push it down to the bottom of the 50mL conical tube. Then, close the cap of the conical tube firmly.

SECTION: COLLECTING THE LYSATE
24. Put the conical tubes in the Megafuge adapters. 
Centrifuge the conical tube containing the Sterivex filter unit at 6,000 x g for 1min and collect the extracted DNA in a 2 mL tube.

SECTION: COLLECTING THE LYSATE
25. Remove the 50 mL conical tube from the centrifuge and remove the Sterivex filter unit and 2.0 mL tube using tweezers.
Note: The 2.0 mL tube is uncapped; handle it carefully. Wipe down the tweezers with PREempt wipes between samples.
SECTION: COLLECTING THE LYSATE
26. Discard the used conical tubes (keep one conical tube and use it as a provisory waste container) and Sterivex filter unit. 
Firmly cap the 2.0 mL tube and proceed with these vials and lysate
SECTION: FINAL INCUBATION AND PRECIPITATION
27. Add 4 μL of RNAseA (100 mg/mL). 
Mix pipetting up and down (or vortexing). 
Incubate at room temperature for 2 min.

SECTION: FINAL INCUBATION AND PRECIPITATION
28. Add 200 μL buffer AL. 
Mix thoroughly pipetting up and down (or by vortexing). It may form some precipitate. 
Incubate at 56°C for 10 min (it doesn’t need to be in the rotisserie).

SECTION: FINAL INCUBATION AND PRECIPITATION
29. Add 200  μL ethanol (96-100%). 
Mix thoroughly pipetting up and down (or by vortexing). 
Total volume now is ~1.2 mL.
SECTION: DNA BINIDNG AND WASHING
30. Pipette 600 μL the mixture into a DNeasy mini spin column placed in a 2 mL collection tube. 
Place the tubes in the centrifuge and spin the tubes at 8,000 rpm (~6,000 x g) for 1 min. 
Discard the flow through.
SECTION: DNA BINIDNG AND WASHING
31. Repeat the previous step once more,  using the remaining volume of the lysate.
  

SECTION: DNA BINIDNG AND WASHING
32. Place the spin column in a new 2 mL collection tube. If after spinning down you still can see some buffer in the spin column, centrifuge the tube again with a higher speed (about 10,000 x g). If this still not work, spin down with a higher speed.
Add 500 μL buffer AW1. 
Centrifuge for 1 min at 8,000 rpm (~6,000 x g). 
Discard the flow through and collection tube.
SECTION: DNA BINIDNG AND WASHING
33. Place the spin column in a new 2 mL collection tube. Add 500 μL buffer AW2. 
Centrifuge for 30 s. at 14,000 rpm (~20,000 x g). 
Discard the flow through and collection tube.
SECTION: DNA BINIDNG AND WASHING
34. Transfer the spin column to a new 1.5 mL or 2 mL microcentrifuge tube.
SECTION: PCR INHIBITOR REMOVAL
36. Zymo-Spin‱ III-HRC Columns need to be prepared prior to use:
 Insert column into a Collection Tube.  *Please note that the matrix in the column may appear dehydrated,
or powdery. This is normal.
Open the cap, add 600 µl of Prep-Solution, and centrifuge at 8,000 x g for 3 minutes.
SECTION: PREPARATION
3. UV for 30 minutes the following:  
Silica beads - you will need a 5mL tube full of beads to extract DNA from 12 samples 
(See bench top guide in the end of this protocol to check the amount of tubes you will need)
2mL tubes
50mL falcon tubes
Racks
Pipettes 
pipette tips

Do not forget to UV tubes for that will be used to aliquot the buffers, and silica beads. 
SECTION: PREPARATION
2. 
 

Materials needed:
  
0.5 mm glass beads, silica. Molecular grade
Molecular grade ethanol (100%)

  
The kit Includes:
ATL (lysis buffer)
AL (lysis buffer)
RNAse
AW1 (Wash buffer)
AW2 (Wash buffer)
AE (Elution buffer)
Spin Columns

  
The kit Includes:
ZYMO spin columns
Prep solution

SECTION: DNA BINIDNG AND WASHING
35. Elute the DNA by adding 100 μL buffer AE to the centre of the spin column membrane. 
Incubate for 1 min. at room temperature. 
Centrifuge for 1 min at 8,000 rpm (~6,000 x g).
SECTION: PCR INHIBITOR REMOVAL
37. Transfer the prepared column to a clean, labeled 1.5 ml microcentrifuge tube.
Add the eluted DNA from    to the Zymo-Spin‱ III-HRC Column.
Centrifuge at 16,000 x g for 3 minutes.
SECTION: PCR INHIBITOR REMOVAL
38. Store the filtered DNA in -80.