Coral Resilience:Notebook/Diagnostic Gene Expression Biomarkers from Montipora capitata/2022/05/05

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Using TNES-6U to extract DNA from Trizol samples Yesterday I made the TNES-6U buffer and added 350 ul of it to Trizol extracts that had their RNA containing aqueous layer removed. I shook the tubes vigorously for ~15 seconds, let them sit at room temp for 10 minutes, and centrifuged them at 12,000g for 20 minutes at 4C. Thereafter, I removed 350 ul of the new aqueous layer and transferred it to new tubes. The remaining phenol was labeled as #_6 P (for protein) and stored at -80C. To the DNA containing tubes, I added an equal volume of 350 ul Isopropanol, vortexed them, and placed them in the -80C overnight. Today I will continue extracting the DNA by following the following protocol: Spin at 12,000xg for 20 min at 4C then discard the supernatant Wash the pellet 3 times with 75% ethanol (incubate 3 min, spin 12,000xg for 5 min) After the third wash, remove most of the remaining ethanol with a gel loading tip. Air dry on bench for 3-10 min or until liquid is not visible