DNA Extraction from Ethanol Zooplankton Samples - Phenol-Chloroform

Materials needed (OR similar):

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.

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.

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.

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.

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.

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.

Add another 1mL sucrose lysis buffer (SLB) to the lysate tube.

• 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.

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).

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).

Add 2 mL TE buffer to Amicon and spin at 3500 g for 6 min. * Remove filtrate.

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.

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.

Quantify DNA stock using Qubit (following manufacturer’s instructions). Use 2 µL of stock DNA when quantifying.

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

Place the 2.5 ng/μL DNA stock in the -20°C freezer.