SRM Dilution Calculations 2
Revised for the last time
Today I went to UWPR to make the dilutions. Last night, Laura told me that she diluted our PRTC to 200 fmol/µL, so I had to account for that when making dilutions. The first thing I did was revise my dilution calculations with the new PRTC stock concentration. Because I needed to add more PRTC to each sample, I changed the injection volume from 1 µL to 2 µL for the 10:1 dilution. This means Laura has to pipet 0.375 µL of geoduck, which is less than 0.5 µL. Because Laura and I have pipetted this volume before and it’s only for one sample, I think it is okay. Before I went down, I took 0.1 µL and 0.5 µL pipets from the lab.
Table 1. Revised 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 | 2 | 0.9 | 0.1 | 6.75 | 0.375 | 1.875 | 6 | 15 |
| 2 | 7.5:1 | 1 | 2 | 0.87 | 0.13 | 6.5 | 0.5 | 1.875 | 6.125 | 15 |
| 3 | 5:1 | 1 | 2 | 0.8 | 0.2 | 6 | 0.75 | 1.875 | 6.35 | 15 |
| 4 | 2.5:1 | 1 | 2 | 0.6 | 0.4 | 4.5 | 1.5 | 1.875 | 7.125 | 15 |
| 5 | 1:2.5 | 1 | 2 | 0.4 | 0.6 | 3 | 2.25 | 1.875 | 7.875 | 15 |
| 6 | 1:5 | 1 | 2 | 0.2 | 0.8 | 1.5 | 3 | 1.875 | 8.625 | 15 |
| 7 | 1:7.5 | 1 | 2 | 0.13 | 0.87 | 1 | 3.25 | 1.875 | 8.875 | 15 |
| 8 | 1:10 | 1 | 1 | 0.1 | 0.9 | 1.5 | 6.75 | 3.75 | 3 | 15 |
I then created 25 µL of my pooled oyster solution by adding 5 µL of the samples I selected here.
- O14
- O39
- O96
- O124
- O137
Using the volumes in Table 1, I prepared the vials by adding my oyster sample, PRTC and ACN. When I got to Vial 6, it was difficult for me to pipet the 1.5 µL I needed, so I made 10 µL of the oyster pool. I finished up the vials and left in the freezer for Laura to add her samples to.