TITLE: DNA Extraction from 0.22µm Sterivex Filters - Phenol-Chloroform
AUTHORS: Colleen Kellogg

[DESCRIPTION]
This protocol is used to extract genomic DNA from 0.22μl sterivex filters using Phenol-Chlorophorm. As part of the Hakai Institute Ocean Observing Program, biomolecular samples have been collected weekly, from 0 m to near bottom (260 m), to genetically characterize plankton communities in the Northern Salish Sea since 2015. 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 Minimum Information about an Omics Protocol (MIOP) and other recommendations under the "Guidelines" tab.

[GUIDELINES]
  MIOP: Minimum Information about an Omics Protocol
 
 
 
 
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RELATED PROTOCOLS
 
 
ACRONYMS AND ABBREVIATIONS
 
 
GLOSSARY
 
 

BACKGROUND

This protocol is used to extract genomic DNA from 0.22μl sterivex filters using Phenol-Chlorophorm.

Spatial coverage and environments of relevance
As part of the Hakai Institute Ocean Observing Program, biomolecular samples have been collected weekly, from 0 to near bottom (260 m), to genetically characterize plankton communities in the Northern Salish Sea since 2015, developing a climatology from which we can begin uncover the physical, chemical and biological drivers of community and functional change in the dynamic coastal waters of coastal British Columbia. 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.

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: LYSIS AND INCUBATION
4. Add 100 µL lysozyme (125 mg fully dissolved in 1000 μl 1 x TE) and 20µL RNase A (10 µg/ml: 1µL in 999µL 1 x TE) to each filter.
Reseal the top with Parafilm or a luer cap.  
Incubate Sterivex in a rotating incubator at 37°C for 1h (in labeled 50 ml falcon tubes).

SECTION: LYSIS AND INCUBATION
5. Add 100 µL Proteinase K and 100 µL (20%) SDS to each filter. 
Reseal using Parafilm or a luer cap.  
Incubate at 55°C for 1-2 hours in a rotating incubator (in 50ml falcon tubes; be sure to keep the same falcon tube for the same sterivex throughout these incubation steps).

SECTION: LYSIS AND INCUBATION
6. Remove lysate from sterivex filter using a 10cc syringe into a labeled 15 ml falcon tube, use a new syringe for each sample. 
 

Repeat as needed until (nearly) all liquid and foam are out of Sterivex. 
Add 1mL sucrose lysis buffer (SLB), rotate Sterivex with your hands several times to rinse the filter out, and then plunge out the liquid as described above. 
Pool this with the lysate in the 15 ml tube.
SECTION: EXTRACTION &amp; CENTRIFUGATION
7. 
In the fume hood, add an equal volume (about 3mL) of Phenol:Chloroform:IAA (25:24:1), pH 8.0 to the lysate tube. 
Invert for 10 secondsby hand to mix.  
Spin at 2500 g for 6 min or until the aqueous layer is clear. Wait at least 10 minutes before opening the centrifuge. 
Transfer the aqueous (top) layer into a new 15 ml falcon tube.
SECTION: EXTRACTION &amp; CENTRIFUGATION
8. Add an equal volume (approx 3mL) of Chloroform:IAA (24:1) to the tube containing the aqueous layer.
Invert for 10 seconds by hand. 
Spin at 2500 g for 6 min or until the aqueous layer is clear with no debris. Wait at least 10 minutes before opening the centrifuge. 
Transfer the aqueous layer into a labeled Amicon Ultra centrifuge tube (UFC801096, EMD Millipore).
SECTION: EXTRACTION &amp; CENTRIFUGATION
9. Top up Amicon with 1-2mL of 1 x TE buffer. 
Spin at 3500 g for 10-15 minutes.
Check to make sure there is less than 1ml liquid left in Amicon at the end of this (if not, fill up with 2mL 1 x TE and spin again).
SECTION: EXTRACTION &amp; CENTRIFUGATION
10. Add 2 mL TE buffer to Amicon and spin at 3500 g for 6 min. 
Remove filtrate.
SECTION: EXTRACTION &amp; CENTRIFUGATION
11. Repeat Step 8 twice more (total of 3 washes with 2mL TE). 
For the last wash, spin until 200 – 500 μl remain in Amicon (typically 8-10 minutes). 
Note the final volume and transfer to a labeled 1.5 μl Eppendorf tube. 
Rinse Amicon sides with 50 µL of 1xTE and pool with the rest of the sample in a labeled 1.5mL Eppendorf tube.
SECTION: DNA QUANTIFICATION AND STORING
12. If desired, aliquot 50 µL from the final sample volume into a 1.5ml Eppendorf tube to use as working stock and place in a -20°C freezer. 
Place the remaining DNA stock in the -80°C freezer for long-term storage.

SECTION: DNA QUANTIFICATION AND STORING
13. Quantify DNA stock using Qubit (following manufacturer’s instructions).  Use 2 µL of stock DNA when quantifying.

SECTION: DNA NORMALISATION
14. 
 

SECTION: DNA NORMALISATION
15. Make a normalized 2.5 ng/μl DNA stock for PCR : 
Calculate the volume of DNA for 2.5 ng/μL stock (there is a spreadsheet for this)  
 

Calculate the volume of water for 2.5 ng/μL stock 
 

SECTION: DNA NORMALISATION
16. Place the 2.5 ng/μL DNA stock in the -20°C freezer.

SECTION: PREPARATIONS
2. 
 


Materials needed (OR similar):
  
  
  
  
  
  
  
  
  
Sucrose Lysis Buffer (SLB) is prepared as follows:
 

SECTION: PREPARATIONS
3. Thaw filters on ice, if applicable. 
Replace Parafilm on each sample if needed. 
Prepare extraction negative - add 1800 µL of SLB (filter-sterilized and UVed) into a new Sterivex. 
Seal the bottom with parafilm and the top with cap or parafilm, treat the same as the rest of the samples.
SECTION: PREPARATIONS
1. The protocol assumes that water has been collected on a sterivex filter and preserved with Sucrose lysis buffer. See the following protocol: