SRM Dilution Calculations
My prior calculations were incorrect!
As I mentioned previously, Laura and I need to run dilutions of our samples to confirm that as the concentration of protein in our samples decreases, the assay does not detect them as intensely. When I showed Emma my calculations, she suggested making dilutions based on protein concentrations (µg) as opposed to µL of sample added. Here are my final numbers:
Table 1. Dilution calculations
| Vial | Dilution Ratio | µg to Inject | µL to Inject | µg oyster Needed | µg geoduck Needed | µL oyster needed ((µg/µL)x15 µL/1 µg/µL) | µL geoduck needed ((µg/µL)x15 µL/2 µg/µL) | PRTC added | µL ACN | Total Volume (µL) |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 10:1 | 1 | 1 | 0.9 | 0.1 | 13.5 | 0.75 | 1.5 | N/A | 15.75 |
| 2 | 7.5:1 | 1 | 2 | 0.87 | 0.13 | 6.5 | 0.5 | 0.75 | 7.25 | 15 |
| 3 | 5:1 | 1 | 2 | 0.8 | 0.2 | 6 | 0.75 | 0.75 | 7.5 | 15 |
| 4 | 2.5:1 | 1 | 2 | 0.6 | 0.4 | 4.5 | 1.5 | 0.75 | 8.25 | 15 |
| 5 | 1:2.5 | 1 | 2 | 0.4 | 0.6 | 3 | 2.25 | 0.75 | 9 | 15 |
| 6 | 1:5 | 1 | 2 | 0.2 | 0.8 | 1.5 | 3 | 0.75 | 9.75 | 15 |
| 7 | 1:7.5 | 1 | 2 | 0.13 | 0.87 | 1 | 3.25 | 0.75 | 10 | 15 |
| 8 | 1:10 | 1 | 1 | 0.1 | 0.9 | 1.5 | 6.75 | 1.5 | 5.25 | 15 |
For PRTC, our stock concentration volume is 0.5 pmol/µL. I changed the injection volume for the 10:1 and 1:10 dilutions from 2 µL to 1 µL to ensure we were pipetting volumes over 0.5 µL. This made it so that the final volume for the 10:1 injection was 15.75 µL, but Emma said that was okay.
Written on July 20, 2017