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

[DESCRIPTION]
This protocol is used to extract genomic DNA from 0.22μl sterivex filters using Phenol-Chlorophorm. The protocol is part of the Hakai Institute's pipeline to analyze microbial and environmental DNA from seawater samples and is implemented as a standard procedure for ongoing sampling programs.

[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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GLOSSARY
 
 

BACKGROUND

This document describes the required protocol to to filter seawater onto a 0.22 micrometer Sterivex filters using paristaltic pump setup.

Method description and rationale

Thiswater filtration is part of the standard best - practice method for analysing microbial and environmental DNA from seawater samples at the Hakai Institutes Genome Lab. The method is part of a pipeline that includes seawater filtration, DNA extraction, and amplicon sequencing.
 
Spatial coverage and environments of relevance

The protocol has been used to analyze water samples (commonly 2L) from all parts of the water columns, nearshore and offshore stations on the coast of British Columbia, Canad.  The protocol is also designed to target a broad range of organisms and has been used for the downstream analysis of Bacteria, Protists, Zooplankton, and Fish - using 16S rRNA, 18S rRNA, COI and 12S respectively.

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
The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two
peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time.

[STEPS]
SECTION: Lysis and Incubation
2. Thaw filters on ice, if applicable. 
Replace Parafilm on each sample if needed. 
Prepare extraction negative - add 1800 µL of SLB or Longmire’s buffer (filter-sterilized and UVed) into a new Sterivex. 
Seal bottom with parafilm and top with cap or parafilm, treat same as rest of samples.
SECTION: Lysis and Incubation
3. Add 100 µL lysozyme (125 mg fully dissolved in 1000 μl 1 x TE) and 20 µL 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 60 min (in labelled 50 ml falcon tubes).

SECTION: Lysis and Incubation
4. Add 100 µL Proteinase K and 100 µL 20 Mass / % volume 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
5. Remove lysate from sterivex filter using a 10cc syringe into a labeled 15 ml falcon tube, use a new syringe for each sample. 
Do not pull up on theplunger while sterivex is attached. You will likely need to do multiple plunges to completely empty the Sterivex, so unscrew the syringe from the filter, pull back the plunger, reattach to Sterivex, and push and hold to plunge liquid out of Sterivex. 
Repeat as needed until (nearly) all liquid and foam are out of Sterivex. 
Add 1 mL sucrose lysis buffer (SLB), rotate Sterivex with your hands several times to rinse the filter out and then plunge out liquid as described above. 
Pool this with the lysate in the 15 ml tube.
SECTION: EXTRACTION &amp; CENTRIFUGATION
6. 
 

In the fume hood, add an equal volume (about 3 mL) 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 aqueous (top) layer into a new 15 ml falcon tube.
SECTION: EXTRACTION &amp; CENTRIFUGATION
7. Add an equal volume (approx 3 mL) 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 aqueous layer is clear with no debris. Wait at least 10 minutes before opening the centrifuge. 
Transfer aqueous layer into a labeled Amicon Ultra centrifuge tube (UFC801096, EMD Millipore).
SECTION: EXTRACTION &amp; CENTRIFUGATION
8. Top up Amicon with 1-2 mL of 1 x TE buffer. 
Spin at 3500 g for 10-15 minutes. 3500 x g 
Check to make sure there is less than 1ml liquid left in Amicon at the end of this (if not, fill up with 2 mL 1xTE and spin again).

SECTION: EXTRACTION &amp; CENTRIFUGATION
9. Add 2 mL TE buffer to Amicon and spin at 3500 g for 6 min. 
Remove filtrate.
SECTION: EXTRACTION &amp; CENTRIFUGATION
10. Repeat Step 8 twice more (total of 3 washes with 2 mL 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 therest of the sample in the a labelled 1.5mL Eppendorf tube.
SECTION: DNA QUANTIFICATION AND STORING
11. If desired, aliquot 50 µL from the final sample volume into a 1.5ml eppendorf tube to use as working stock and place in -20 °C freezer. 
Place the remaining DNA stock in the -70 °C freezer for long term storage.

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

SECTION: DNA NORMALISATION
13. 
 

SECTION: DNA NORMALISATION
14. Make a normalized 2.5 ng/μl DNA stock for PCR : 
Calculate volume of DNA for 2.5 ng/μL stock (there is a spreadsheet for this)  
 
Where [DNA] is the DNA concentration from Qubit and x is the number of uL of the DNA stock that you need to add for a 25 uL volume of a 2.5 ng/uL DNA stock. 
Calculate volume of water for 2.5 ng/μL stock 
  
 

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

SECTION: Preparations
1. 
 

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 top with cap or parafilm, treat thesame as therest of samples.