Acropora hyacinthus eggs and sperm from FACE PUF Morea Project

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For detailed extraction protocol view post: Protocol for DNA/RNA Extractions of Montipora Coral Larvae Using Zymo-Duet Extraction Kit

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Extraction Date: August 20, 2019

Samples

Extraction Number	Sample ID	Content	Sample Quantity (µL)	Sample Conserv.	Tech
21	014	Sperm	100	300 uL Shield	Erin
22	755	Sperm	100	300 uL Shield	Erin

Extraction Notes

• Altough samples were already preserved in 300µL DNA/RNA Sheild, I added an additional 300µL of shield to prevent RNA/DNA degradation during digest
• After adding 300µL shield I transferred the samples to a 1.7mL centrifuge tube in order to digest for 1.5 hours, 55°C, 1100 rpm
• Added additional 15µL Proteinase K after 2 hours
• To accomodate the use of extra shield, we added 750µL Lysis buffer instead of 345µL (Step 3 DNA Extraction). Additionally, we mixed 1000µL EtOH instead of 700µL in 5µL tubes (Step 1 RNA Extraction) and repeated steps 6-7 once.

Qubit

• Followed Broad Range dsDNA and RNA Qubit protocol
• All samples were read twice

Extraction #	Sample ID	DNA Standard 1 (RFU)	DNA Standard 2 (RFU)	DNA 1 (ng/µl)	DNA 2 (ng/µl)	Average DNA	RNA Standard 1 (RFU)	RNA Standard 2 (RFU)	RNA 1 (ng/µl)	RNA 2 (ng/ul)	Average RNA
21	14	164.0	18126	60.8	60.6	60.7	390.6	9983	Too Low	Too Low	Too Low
22	755	164.0	18126	47.0	46.8	46.9	390.6	9983	34.4	34.4	34.4

Tape Station

• Followed RNA TapeStation protocol
• Only Tape Stationed 755

Gel

• Followed PPP Lab Agarose Gel protocol for a medium 1% agarose gel
• Used the Thermo Scientific GeneRuler 1 kb Plus DNA Ladder (link)

Notebook Entry 20190820