Genomic DNA extraction from the diatom Pseudo-nitzschia multistriata for Illumina sequencing

Grow cells as described in: dx.doi.org/10.17504/protocols.io.bgudjws6

Detect the presence/absence of bacteria and collect the cultures as described in: dx.doi.org/10.17504/protocols.io.btt5nnq6

Resuspend cells with 500 μL of TE buffer (10 mM TrisHCl pH 7.6 and 1 mM EDTA pH 8.0)

Add:

• 400 mg of 0.2-0.3 mm diameter zirconia/silica beads
• 500 μL phenol (pH 7.8).

Mix with vortex 3 times at 30 Hz for 85 seconds (Each time put sample in ice for 60 seconds before vortex)

Centrifuge at 11000 g for 5 minutes at 4°C.

Recover aqueous phase in new 1.5 mL Eppendorf tubes (about 600 μl)

Add 500 μL of P.C.I (Phenol:Chloroform:Isoamyl alcohol 25:24:1 v/v) and mix by inversion

Centrifuge at 11000 g for 5 minutes at 4°C

Move the aqueous phase in a new Eppendorf tube and add 5 µL of RNase-A 10 mg/mL

Incubate at 37 °C for 30 minutes.

Add 500 μl di P.C.I (Phenol:Chloroform:Isoamyl alcohol 25:24:1 v/v) and mix by inversion.

Centrifuge at 11000 g for 5 minutes at 4°C.

Move the aqueous phase in a new 2 mL Eppendorf tube and add:

• 50 μL of 3 M NaAc (pH ± 5)
• 1 mL of ethanol 96% (- 20 °C)
• 2 μL glycogen (- 20 °C)

Incubate over night at -20°C.

Centrifuge the samples at 13000 g for 30 minutes at 4°C

Discard aqueous phase

Add 1 mL ethanol 70% and mix gently by inversion

Centrifuge at 13000 g for 10 minutes at 4°C

Discard aqueous phase

Add 1 mL ethanol 70% and mix gently by inversion

Centrifuge at 13000 g for 10 minutes at 4°C

Remove aqueous phase and dry pellet at R.T. for at least 20 minutes

Add 50 μL of preheated (55°C) TE 1X (pH 8) or sterile MilliQ water

Incubate at 55 °C for 20 minutes

Quantify DNA concentration by Nanodrop or Qubit

Check DNA integrity.

Run a small amount of DNA with 1% agarose gel

Store DNA at +4 °C, or -20°C for longer storage times