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 .
