Doing qPCR for Viral Titre on a Test Subset of Samples From Freezing Experiment 1
The freezing experiments consist of infecting flies with DiNV, and freezing them on day 0, 3, 5, and 10. From the PCR data, a lot of samples ended up having what looks like contamination in some way, where the sterile poke flies have virus PCR products. Even when looking at the band strength of those PCRs, some seem pretty strong. However, it is hard to tell exactly what is going on with conventional PCR, so I chose 3 samples from each treatment from each day (24 total) to do qPCR on. Those samples are chosen in the band strength notebook.
Before qPCR, all samples were Qubited and diluted to a 1ng/ul concentration using DNA hydration solution. The Qubit protocol was used, and all samples were thawed and kept on ice during.
| sample_ID | qubit | vol DNA needed | vol hydration solution needed for 1ng/ul |
|---|---|---|---|
| 1 | 31.3 | 3 | 90.9 |
| 9 | 109 | 3 | 324 |
| 10 | 45.8 | 3 | 134.4 |
| 23 | 30.6 | 3 | 88.8 |
| 30 | 44.7 | 3 | 131.1 |
| 16 | 29.5 | 3 | 85.5 |
| 37 | 123 | 3 | 366 |
| 32 | 40.3 | 3 | 117.9 |
| 38 | 55.5 | 3 | 163.5 |
| 42 | 18.3 | 3 | 51.9 |
| 48 | 70.1 | 3 | 207.3 |
| 64 | 67.6 | 3 | 199.8 |
| 84 | 38.4 | 3 | 112.2 |
| 87 | 102 | 3 | 303 |
| 89 | 103 | 3 | 306 |
| 94 | 33.9 | 3 | 98.7 |
| 98 | 43.8 | 3 | 128.4 |
| 117 | 51.3 | 3 | 150.9 |
| 142 | 30.9 | 3 | 89.7 |
| 147 | 65.4 | 3 | 193.2 |
| 143 | 37.6 | 3 | 109.8 |
| 150 | 50 | 3 | 147 |
| 158 | 37 | 3 | 108 |
| 173 | 44.3 | 3 | 129.9 |
Here is the qPCR layout:
- Samples and reagents were thawed on ice, vortexed, and spun down before use
- There were 72 individual wells for each primer, 3 were added on for pipetting error
- 115 master mix:
- 5ul Sso supermix * 75 = 375ul
- 0.5ul 115 F primer * 75 = 37.5ul
- 0.5ul 115 R primer * 75 = 37.5ul
- 3ul nuclease free water * 75 = 225ul
- The master mix was made on ice, vortexed, and spun down
- TPI master mix:
- 5ul Sso supermix * 75 = 375ul
- 0.5ul TPI F primer * 75 = 37.5ul
- 0.5ul TPI R primer * 75 = 37.5ul
- 3ul nuclease free water * 75 = 225ul
- The master mix was made on ice, vortexed, and spun down
- I used the qPCR specific plates and seals
- 9ul of the appropriate master mix was added to the planned well (see layout above)
- 1ul of DNA was added to the planned well (see table above)
- Each well was pipette mixed with 5ul using a multichannel
- The plate was sealed and centrifuged for 2 minutes at 3,000g
- The plate was put in the qPCR machine:
- Used program CFX manager
- Selected user-defined protocol, existing protocol, and KMM folder
- Selected the p47 program
- Used the machine buttons to open and close the lid
- Started the program
- Data from the qPCR machine can be found here
- And analysis of the Cq results can be found here