TITLE: DNA Extraction from Ethanol Zooplankton Samples - Phenol-Chloroform
AUTHORS: Andreas Novotny, Colleen Kellogg, rute.carvalho Carvalho, Matt Lemay

[DESCRIPTION]
This protocol extracts genomic DNA from bulk zooplankton samples, preserved either in ethanol or frozen - using Phenol/Chloroform extraction. The protocol was developed to be as similar as possible to the standard protocol used for environmental DNA at Hakai Institute Ocean Observing Program, to be used for downstream DNA metabarcoding:
 


[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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ACRONYMS AND ABBREVIATIONS
 
 
GLOSSARY
 
 

BACKGROUND

This protocol extracts genomic DNA from bulk zooplankton samples, preserved either in ethanol or frozen - using Phenol/Chloroform extraction. The protocol was developed to be as similar as possible to the standard protocol used for environmental DNA at Hakai Institute Ocean Observing Program, to be used for downstream DNA metabarcoding:
 
Spatial coverage and environments of relevance
As part of the Hakai Institute Ocean Observing Program, zooplankton samples have been collected monthly to biweekly, using a 250um bongo net with an opening diameter of 0.5m, towed from 230m-0m. The zooplankton samples have been preserved in formalin for microscopic analysis and in 99% ethanol for genetic analysis.

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
6. 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 sample tube.
Incubate samples in a rotating incubator at 37°C for 1h.

SECTION: LYSIS AND INCUBATION
7. Add 100 µL Proteinase K and 100 µL (20%) SDS to each lysate tube. 
Incubate at 55°C for 1-2 hours in a rotating incubator.

SECTION: LYSIS AND INCUBATION
8. Add another 1mL sucrose lysis buffer (SLB) to the lysate tube.
SECTION: EXTRACTION &amp; CENTRIFUGATION
9. 
 

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
10. 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
11. 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
12. Add 2 mL TE buffer to Amicon and spin at 3500 g for 6 min. 
Remove filtrate.
SECTION: EXTRACTION &amp; CENTRIFUGATION
13. 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
14. 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
15. Quantify DNA stock using Qubit (following manufacturer’s instructions).  Use 2 µL of stock DNA when quantifying.

SECTION: PREPARATIONS
1. 
 

Materials needed (OR similar):
  
  
  
  
  
  
  
  
  
Sucrose Lysis Buffer (SLB) is prepared as follow:
 
To be Autoclaved:
  
  
SECTION: ZOOPLANKTON HOMOGENIZATION
2. Subset the bulk zooplankton sample to a to a reasoable volume (here, 1/8th of the original sample):
Use a plankton net filter to remove the ethanol preservative (keep the preservative).
Resuspend zooplankton in dH2O
Use a Fulton splitter to subset 1/8 fraction of the zooplankton sample and add to a 50ml falcon tube.
Return the remaining fractions to the ethanol preservative.
SECTION: ZOOPLANKTON HOMOGENIZATION
3. Centrifuge the falcon tube 5000xg for 5 min to pellet the zooplankton
Use a pipett to remove all liquid.
Add SLB to the falcon tube, to a total volume of 6mL, and resuspend.
SECTION: ZOOPLANKTON HOMOGENIZATION
4. Add 3 large UFO beads (3.5 mm) to the falcon tube.
Add ~30 small stainless steal beads (0.9-2.0 mm) to the falcon tube.
Vortex vigorously at full speed for at least 10 min, or until no visible structures of the zooplankton are left.
SECTION: ZOOPLANKTON HOMOGENIZATION
5. For each sample, add 1800 µL SLB to new labeled 15mL falcon tubes.
Add an aliquot of 100 µL of the homogenized zooplankton sample to the SLB tube. The total volume should be around 1900 µL.
SECTION: DNA NORMALISATION
18. Place the 2.5 ng/μL DNA stock in the -20°C freezer.

SECTION: DNA NORMALISATION
17. 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.