version: April 06, 2020

About this document

This protocol is used to extract high quality genomic DNA from both coral host and Symbiodiniaceae spp. The resulting DNA should be sufficient for downstream PCR and high-thoughout sequencing. This protocol was utilized in Eckert, R. J., Studivan, M. S., and Voss, J. D. (2019) Populations of the coral species Montastraea cavernosa on the Belize Barrier Reef lack vertical connectivity. Sci. Rep. 9, 7200. doi:10.1038/s41598-019-43479-x to examine coral host population genetic structure.

Additionally, the DNA extraction and clean-up were used in Eckert RJ, Reaume AM, Sturm AB, Studivan MS and Voss JD (2020) Depth influences Symbiodiniaceae associations among Montastraea cavernosa corals on the Belize Barrier Reef. Front. Microbiol. 11:518. doi: 10.3389/fmicb.2020.00518. to examine Symbiodiniaceae community structure across depth through ITS2 metabarcoding.

CTAB gDNA Extraction Protocol

Revised from Mieog et al. (2009) and http://ccb.ucr.edu/lab_protocols.html

Supplies

The following supplies are necessary to perform a round of 24 extractions.

Supplies Reagents Equipment
72 2 mL tubes (3 sets of 24) 2X CTAB Extraction Buffer 4 ˚C centrifuge
24 scalpel or razor blades Proteinase K (20 mg/mL) Bead homogenizer
Parafilm Chloroform:Isoamyl Alcohol (24:1) Thermomixer
0.5 mm glass beads Isopropanol @ -20 ˚C Nanodrop
Kimwipes 70% Ethanol
1X TE Buffer pH 8.0

Reagent recipes

Recipes for making ectraction buffer and associated stock solutions necessary for CTAB gDNA extraction follow.

2X CTAB extraction buffer

Use heat and stirring to dissolve CTAB into solution before adding NaCl. Make CTAB buffer just prior to extractions. Can keep at room temperature for several days.
Reagent per 20 mL Target
CTAB 0.4 g 2%
1M Tris-HCl (pH 8.0) 2.0 mL 100 mM
0.5M EDTA (pH 8.0) 800 µL 20 mM
5M NaCl (add after CTAB dissolves, but before DEPC Treated H2O) 5.6 mL 1.4 M
DEPC Treated H2O to 20.0 mL
20 mg/mL Proteinase K (Do not add to buffer) 0.8 µL per sample 20 µg/mL

Reagent stock recipes

Reagent per 100 mL
5M NaCl 29.22 g NaCl + 80 mL DEPC-treated H2O. Add DEPC Treated H2O to 100 mL.
1M Tris-HCl (pH 8.0) 12.11 g Tris + 80 mL DEPC-treated H2O. Adjust pH with HCl and add DEPC-treated H2O to 100 mL.
0.5M EDTA (pH 8.0) 18.61 g EDTA + 80 mL DEPC-treated H2O. Adjust pH with NaOH and add DEPC-treated H2O to 100 mL.
1X TE Buffer (pH 8.0) 1 mL 1M Tris-HCl pH 8 +200 µL 0.5M EDTA pH 8.0. Add DEPC-treated H2O to 100 mL.

Genomic DNA Extraction

Set heatblock to 55 ºC
Set refridgerated centrifuge and set to 4 ºC

  1. Prepare CTAB extraction buffer just prior to use, do not add proteinase K.
  2. Scrape tissue from coral fragment and place into a 2 mL tube with 0.1 mL (~ 0.075 g) of 0.5 mm glass beads.
  3. Add 800 µL CTAB extraction buffer.
  4. Add 0.8 µL Proteinase K. Seal tubes with parafilm. Invert to mix.
  5. Bead beat for 2–3 mins (6 M/s, three 45 sec intervals w/ 2 min cool down between).
  6. Incubate at 60 ºC for 90 min while mixing.
  7. Add 800 µL Chloroform:Isoamyl Alcohol (24:1). Invert to mix.
  8. Mix and centrifuge at 20,000 x g for 15 mins at 4 ºC.
  9. Transfer aqueous phase to new 2 mL tube (600 µL then 150 µL), taking care not to disturb interphase layer.
  10. Add 800 µL cold (-20 ºC) Isopropanol.
  11. Mix and incubate for 20 min at -20 ºC.
  12. Centrifuge at 20,000 x g for 20 min at 4 ºC.
  13. Carefully pour off supernatant.
  14. Add 150 µL of 70% Ethanol at room temperature. Invert to mix.
  15. Centrifuge at 20,000 x g for 5 min at 4 ºC.
  16. Remove supernatant (pour off, quick spin, pipette off remaining avoiding pellet) and dry inverted on Kimwipe for 15 min at room temperature.
  17. Elute in 100–200 µL of 1X TE pH 8.0.
  18. Incubate at 55 ºC for 10 min.

Cleaning genomic DNA

After extracting genomic DNA, use Zymo DNA Clean & Concentrator-5 (D4014) to clean DNA and remove inhibitors prior to running a PCR. This greatly improves nanodrop 230/260 and 260/280 readings, dramatically increasing amplification success.

Prior to first use add ethanol to Wash Buffer and label bottle.

  1. Measure DNA concentration (Nanodrop okay for this step). Prepare 0.6 mL tubes with 5µg (or less) DNA in 100 µL total volume 1X TE to be cleaned.
  2. Set Elution Buffer for elution step in heat block at 60–70 ºC.
  3. In a 2 mL tube add 2:1 volume of Binding Buffer:DNA (200 µL) to each volume of genomic DNA and vortex thoroughly.
  4. Transfer the mixture to a provided Zymo-Spin Column in a collection tube.
  5. Centrifuge 10,000 x g for 30 sec at room temperature. Discard flow through.
  6. Add 200μL DNA Wash Buffer to the column. Centrifuge at 10,000 x g for 1 min at room temperature. Repeat.
  7. Transfer the column to a new labeled 1.5 mL tube. Elute by adding 20 µL of Elution Buffer directly to the column matrix and incubate at room temperature for 3–5 min. Centrifuge for 30 seconds to elute DNA.
  8. Check DNA quality with Nanodrop and quantify DNA flourescently (e.g. Qubit) and prepare 10 ng/µL dilutions.