Bulk gDNA extraction from coral samples
Acquire sample material.
Small amounts of coral tissue, with or without skeleton, may be obtained by clipping off branch tips, scraping with a razor blade, etc.
Add sample to 500 µL 1% SDS in DNAB in a microcentrifuge tube.
Be sure that sample is fully immersed in the buffer.
Incubate sample for 60-90 minutes at 65°C.
Sample is now stabilized for storage at room temperature, and can be treated as an "archive" for future use.
These archives can be used for multiple attempts at DNA extraction.
Add 25 µL Proteinase K (10 mg/mL) to sample archive and vortex well.
Incubate overnight at 37°C, for 6-7 hours at 45°C, or for 2-3 hours at 55°C.
Prepare a new set of 1.5 mL tubes for the samples you intend to process, and add 100 µL of each sample archive (in 1% SDS in DNAB) to the new set of tubes.
Return the remainder of the sample archive to storage.
Defrost CTAB mix (stored at -20°C) and add twice volume (200 µL) to each sample.
Vortex and incubate at 65°C for 30-60 minutes.
Allow samples to cool.
In fume hood, add equal volume (300 µL) of chloroform.
Be sure to ‘charge’ (i.e., fill and empty pipette tip with chloroform 2 to 3 times) the pipette tip before first use, or your tip will leak chloroform.
Vortex sample and invert several times, but be careful that caps are tight – leaking chloroform will erase your sample labels!
Put in rack on rotating platform for 2-3 hours.
Centrifuge at 10,000g (RCF) for 10 minutes.
Align tubes in centrifuge so that hinges are on the outside.
While spinning, prepare a new set of labeled 1.5 mL tubes.
Remove samples from centrifuge and very carefully pipette off top ~250 µL into new tube.
Dispose the rest of the contents into appropriate waste container.
Add twice volume (500 µL) of 100% (200-proof) ethanol (EtOH).
Ensure caps are shut tightly and invert samples in their rack several times, together with a few brief shakes to make sure samples are well mixed.
Put samples in freezer for at least 2 hours to promote DNA precipitation.
If the EtOH is pre-chilled, you can leave it in the -20°C freezer for only a 1/2 hour.
Put samples in centrifuge (ensuring that the hinges of the tubes are on the outside) and spin for 10 minutes at 10,000g (RCF).
Remove samples from centrifuge and carefully decant off ethanol from all the tubes into a waste container.
The DNA pellet should remain stuck to the inside of the tube.
Put tubes, with their caps open, in the Vacufuge/Speedvac.
Be careful when putting the tubes in and don’t touch the inside of the caps.
Speedvac at 45°C for 30-60 minutes.
Remove samples from vacufuge and add 100 µL of 0.3 M NaOAc (do not use the stock 3 M solution!).
Vortex sample well to dissolve pellet.
When the pellet is dissolved the sample will appear “syrupy” and will not bounce around as droplets inside the tube.
Once the pellet is dissolved, add 200uL of 100% Ethanol, vortex and invert several times and put in freezer for at least 2hrs.
Remove samples from freezer, and centrifuge for 10 minutes at 10,000g (RCF).
Decant supernatant into appropriate waste container.
Add 100 µL of 70% Ethanol, and vortex thoroughly (this is the “Ethanol Wash” step).
Centrifuge for 10 minutes at 10,000g (RCF), and again decant supernatant into appropriate waste container.
Put samples in Vacufuge with the caps open, and speedvac at 45°C for 30-60 minutes to thoroughly dry the pellet.
Take samples out of centrifuge and add 50-100 µL TE buffer.
Vortex briefly to mix and store at -20°C in freezer.
Sample is now ready for PCR.
Store DNA samples at -20°C.
