Sep 30, 2017
Modified genomic DNA extraction method for Heterosigma akashiwo
- Monica Accerbi1,
- Vinay K Nagarajan1,
- Kathryn Coyne2,
- Pamela J Green1
- 1Delaware Biotechnology Institute, University of Delaware;
- 2School of Marine Science and Policy, University of Delaware
Protocol Citation: Monica Accerbi, Vinay K Nagarajan, Kathryn Coyne, Pamela J Green 2017. Modified genomic DNA extraction method for Heterosigma akashiwo. protocols.io https://dx.doi.org/10.17504/protocols.io.himb4c6
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
Created: March 31, 2017
Last Modified: March 22, 2018
Protocol Integer ID: 5421
Keywords: genomic, DNA, CTAB, Heterosigma
Abstract
This protocol was developed for isolating high quality genomic DNA from Heterosigma akashiwo for the purpose of Next generation sequencing technologies. We successfully repeated this protocol to obtain genomic DNA.
Starting material: Start with 2 x 100 ml axenic culture, about 2.5 x 105 cell/ml
Divide into 50 ml round bottom tubes and centrifuge in a swing rotor at 2,000 rpm for 2’
Pour off liquid and add 2 ml of DNA Extraction Buffer per tube
DNA Extraction Buffer
(final concentrations are shown)
CTAB (hexadecyltrimethylammonium bromide) 2% (w/v)
Tris Buffer pH 8.0 100 mM
EDTA (Ethylenediaminetetraacetic acid) 20 mM
NaCl 1.4 M
PVP (Polyvinylpyrrolidone, MW 40,000) 1% (w/v)
Prepared in DNase/RNase-free water
Add 2% (v/v) β-mercaptoethanol to the amount of extraction buffer needed each time just before starting
Note
Prepare all buffers with sterile solutions and bottles so you could skip autoclaving very viscous solutions. CTAB-containing buffers will need overnight stirring for CTAB to dissolve completely
Homogenize [PT3100 Polytron] at max speed 10”-15” to disrupt pellet
Combine homogenate in two 15 ml tubes
Add 8 ml DNA Extraction Buffer to each tube
For the following steps, use gentle inversion to mix. DO NOT vortex!
Incubate at 65°C for 1 h gently mixing every 10’
Add RNase A to a final concentration of 100 µg/ml, and incubate at 37°C for 30’
Centrifuge at 13,000 rpm, at room temperature for 10’
Transfer supernatant to a new tube, extract with same volume of phenol:chloroform:isoamyl alcohol (IAA) (25:24:1, pH6.6), and centrifuge at 13,000 rpm for 2’ at 4°C
Transfer the aqueous upper phase and add 1/10 volume of pre-warmed 65°C High Salt Buffer and mix well by inversion
High Salt Buffer
(final concentrations are shown)
CTAB 10% (w/v)
NaCl 0.7 M
Prepared in DNase/RNase-free water
Note
CTAB is added to the above solution in batches and allowed to dissolve overnight
Extract with same volume of phenol:chloroform:IAA as in step 10
Transfer the aqueous upper phase, and extract with same volume of chloroform:IAA (24:1); should have pretty clean aqueous/organic interface at this step
Transfer the aqueous upper phase into a new tube, add 0.8 volume of isopropanol and 40 µg of glycoblue
Precipitate genomic DNA at -80°C overnight
Centrifuge at 13,000 rpm, 4°C for 30’
Discard supernatant and wash pellet twice with 70% (v/v) ethanol
Air dry the DNA pellet and dissolve it in 200 µl Qiagen elution buffer
Expected yield 25-30 µg