E26, E27, E28, E29, E30, E31, E32, E33

Procedure

added 180 microliters ATL to eight 1.5ml eppendorf tubes Labeled the tubes E26, E27, E28, E29, E30, E31, E32, E33

Took original coral samples of sample E26, E27, E28, E29, E30, E31, E32, E33 from -20° freezer

For each coral: took coral out of tube with sterolized forceps
placed on a sterolized glass slide cut small piece of tissue off with sterlolized scissors placed in the corresponding eppendorf tubes

added 20 microliters of Protinase k to each tube

samples were placed in Thermomixer set to 50°C and 600rpm

Took samples out of thermomixer after 1 hour Vortexted samples after observing the tissue had been dissolved

added 200 microliters Al buffer to each and Vortexted added 200 microliters of 100% ethonol

transfered samples to spin columns centrifuged for 1 minute at 8000 rpm

transfered spin columns to new collection tubes added 500 microliters of buffer AW1 centrifuged for 1 minute at 8000 rpm

transfered spin columns to new collection tubes added 500 microliters of buffer AW2 centrifuged for 3 minutes at 14000 rpm

transfered spin columns to eppendorf tubes added 50 microliters of buffer AE (warmed to 56°C) let liquid incubate for 5 minutes at room temperature centrifuged for 1 minute at 8000 rpm added 30 microliters of buffer AE (warmed to 56°C) incubated for 5 minutes at room temperature centrifuged for 1 minute at 8000 rpm

labeled as E19, E21, E22, E23, E24, E25 placed in box labeled “DNA Extractons Genus: Eunicia” placed box in -20° standup freezer

Additional Notes

This completed the full set of extractions (there is now an extraction of each eunicia sample)

E29 was very clear after dissolving the coral fragment 50 microliters extra AE buffer was added to E29

• This protocol modified from DNeasy Blood and Tissue Handbook