Sep 29, 2021

Public workspaceGenomic DNA extraction from the diatom Pseudo-nitzschia multistriata for Illumina sequencing V.5

DOI
dx.doi.org/10.17504/protocols.io.byk7puzn
  • 1Stazione Zoologica Anton Dohrn;
  • 2SZN
Francesco Manfellotto
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.byk7puzn
Protocol Citation: Francesco Manfellotto, Monia Teresa Russo, Pina Marotta, Rossella Annunziata, Anna Santin, Antonella Ruggiero, Mariella Ferrante 2021. Genomic DNA extraction from the diatom Pseudo-nitzschia multistriata for Illumina sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.byk7puznVersion created by Francesco Manfellotto
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: September 28, 2021
Last Modified: September 29, 2021
Protocol Integer ID: 53631
Abstract
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.
5m
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
5m
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.
30m
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.
5m
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.
12h
Centrifuge the samples at 13000 g for 30 minutes at 4°C
30m
Discard aqueous phase
Add 1 mL ethanol 70% and mix gently by inversion
Centrifuge at 13000 g for 10 minutes at 4°C
10m
Discard aqueous phase
Add 1 mL ethanol 70% and mix gently by inversion
Centrifuge at 13000 g for 10 minutes at 4°C
10m
Remove aqueous phase and dry pellet at R.T. for at least 20 minutes
20m
Add 50 μL of preheated (55°C) TE 1X (pH 8) or sterile MilliQ water

Incubate at 55 °C for 20 minutes
20m
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