E2, E3, E4

Procedure

added 180 microliters ATL to four 1.5ml eppendorf tubes Labeled the tubes E2, E3, E4

Took original coral samples of sample E2, E3, E4 from -20° freezer

For each coral: took coral out of tube 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

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 20 microliters of buffer AE (warmed to 56°C) incubated for 5 minutes at room temperature centrifuged for 1 minute at 8000 rpm

labeled as E2, E3, E4 placed in box labeled “DNA Extractons Genus: Eunicia” placed box in -20° standup freezer

• This protocol modified from DNeasy Blood and Tissue Handbook