DNA extraction from seawater

• Makes sure that screw cap microcentrifuge tubes are available (sterile). I prefer 2.0 ml but 1.5 ml will suffice.
• Solutions, freshly made and autoclaved:

1. TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
2. STE (100 mM NaCl, 10 mM Tris-HCl, 1mM EDTA, pH 8.0)
3. 3 M Sodium Acetate pH 7.0
4. TE saturated Phenol
5. Chloroform:IAA 24:1
6. 20 % SDS

• Glycogen (Molecular Biology Grade)
• Find a working filtration manifold (2) with trap, pump and tubing (2 manifolds can be plumbed to one trap and vacuum pump if you are filtering a lot of samples)

Sample Filtration

• Using a filtration manifold, place a 0.45 Millipore Durapore (25 mm) filter each in two filtration towers. Clamp and suck say 3-5 ml DI water through to make sure that it is not leaking. Do this by turning on the vacuum pump (set at 5-7 PSI vacuum).
• Filter at least 200 ml through each filter. Note, if 200 ml is filtered, then try another 50 ml. Record the volume used-try to filter as much through the filter as feasible up to about 800 ml. This may take 20 min.
• While vacuum is still on, remove the tower, and use the forceps to roll up the filter and place in a 1.5 ml screw cap microfuge tube containing 1 ml of STE. Note-if you were at sea and could not process your sample you could freeze it and extract back at the lab.

• If samples are frozen thaw in warm water-bath.
• Carefully add 0.1 volume of 20% SDS (sodium dodecyl sulfate). This is a detergent that aids in cell lysis.

• Using a styrafoam microfuge “float”, immerse your tube in boiling water for 2.5 minutes.
• Cool on ice for 1 min, then centrifuge for 10 min at top speed.
• Collect the supernatant into a fresh tube and place on ice. Add a second ml of STE and 0.1 volume of 20% SDS to the filter and pellet, vortex, re-boil, spin and recover supernatant.
• Asess lysis by making a slide of the 2nd extract by putting 10 ul on a microscipe slide followed by a cover slip. View the slide under 400X or 1000X and phase contrast. If many whole algal cells are present, than do a third round of boiling for 2.5 min. Otherwise, proceed.
• Combine the supernatants, add 0.1 volume of 3M sodium acetate pH 7 and mix well.
• Add two volumes of 95 or 100% ethanol, vortex to mix, and store at -80oC for 30 min. Note: to simplify and keep everything in microcentrifuge tubes, you may want to aliquot the supernatants (extracts) into 500 ul aliquots and then add 1.0 ml of ethanol. This should take four 1.5 ml centrifuge tubes.
• Centrifuge at top speed, carefully decant off the supernatant and save the pellets. Air dry at 65o in the ‘electric cat box’.
• Take up the pellet in 0.5 ml TE. Do this by adding 0.5 ml to ONE tube only, triturating with a P-1000 pipette. Then transfer the 0.5 ml to the second tube, repeat. Do this until all four tubes have been taken up in the original 0.5 ml volume of TE.
• Add 0.5 ml TE saturated phenol (pH 8.0) and vortex gently
• Centrifuge for 4 min and collect supernatant.
• Extract supernatant with 0.3 ml phenol and 0.3 ml chloroform:isoamyl alcohol (24:1)
• Centrifuge for 4 min; collect the supernatant and repeat the last two steps.
• Extract with 0.5 ml chloroform:isoamyl alcohol (24:1) and collect supernatant.
• Add 1 μl molecular biology grade glycogen. This aids in precipitation. Add 0.1 volumes of 3M sodium acetate pH 7, mix well. Add two volumes of ethanol and incubate @ -80oC for 30 min.
• Centrifuge for 10 min at top speed. Immediately remove tube from centrifuge and mark the outside (where a visible or invisible pellet) may be. Carefully decant the supernatant.
• Add 0.5 ml ice-cold 70% ethanol.
• Centrifuge for 10 min. Discard the supernatant and air dry at 65oC.
• Take up the pellet in 100 μl of TE. Quantify the DNA (2 or 5 μl) using the Hoechst procedure described elsewhere in this manual. Label your tube and freeze in your DNA boxes at –80oC. Frozen this way DNA should outlast your children’s children and your children’s children’s children and ……