E8 E9 E10 E16 E17 E19 E20 E21 E24 E28 E30 E32 P14 P15

• These are the samples that after multiple attempts have not shown up in any ITS2 PCR’s

Procedure

added 180 microliters ATL to 14 1.5ml eppendorf tubes Labeled the tubes acoarding to the code

Took original coral samples of samples 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

then 20 microliters of Protinase k was added 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

P14 and P15 were placed in box labeled “ALiquoted DNA smaples” The Eunicia samples were placed in a box “Eunicia DNA extractions” both boxes were put in the -20° standup freezer

• This protocol modified from DNeasy Blood and Tissue Handbook