Based on Putnam Lab Protocol written by M. Schedl: Qubit

Broad Range dsDNA, Broad Range RNA, and High Sensitivity RNA

All Steps are the same whichever quantification method you are using

1. Determine n number: Number of samples + 2 + % error
2. Create master mix of Qubit Buffer and Quant-IT Reagent
   • n μl of Quant-IT Reagent
   • 199 * n μl of Buffer
3. Vortex and spin down master mix
4. Set up thin walled tubes: enough for each of your samples plus 2 more for standards
5. Add 190μl of master mix to the 2 tubes for the standards
6. Add 199μl of master mix to each of your sample tubes
7. Take standards out of the fridge (they say HP for the Putnam Lab) and add 10μl of Standard 1 to the first standard thin walled tube and add 10μl of Standard 2 to the second standard thin walled tube.
8. Add 1μl of sample to the corresponding thin walled sample tube.
9. Vortex and spin down all tubes.
10. Choose appropriate assay in the Qubit and read standard tubes.
11. If your standard values are not near the range of values in these graphs then your master mix was probably made wrong.
12. Read each sample tube one at a time. Our settings are 1μl of sample and we want the output to be ng/μl.
13. Read your samples twice.
14. All components are safe and can be disposed of in the tip buckets or trash.

Holobiont Integration project Master Mix Calculations:
4 coral fragments (4 soft homogenized, 4 hard homogenized = 8 total):

• 8 samples + 2 standards + 0.2% error = 10.2 µl Quant-IT Reagent
• 199 * 10.2 = 2,029.8 µl Qubit buffer

8 coral fragments (8 soft homogenized, 8 hard homogenized = 16 total):

• 16 samples + 2 standards + 0.2% error = 18.2 µl Quant-IT Reagent
• 199 * 18.2 = 3,621.8 µl Qubit buffer

10 coral fragments (10 soft homogenized, 10 hard homogenized = 20 total):

• 20 samples + 2 standards + 0.2% error = 22.2 µl Quant-IT Reagent
• 199 * 22.2 = 4,417.8 µl Qubit buffer