qPCR to Determine Difference in Viral DNA Yield Between the Hirt and Puregene Extracted Dinn Cells, as Well as Adding in Some D. innubila Adults Who Died from DiNV Infection to See Viral Load
Information on the samples from the Hirt extraction can be found here. Their corresponding Puregene samples were extracted here along with a Qubit to determine DNA concentration. Fly DNA used to check the integrity of replicate 2 of the male and female 16Cq injections, extracted here, was also used in the qPCR.
All DNA samples were thawed on ice, then diluted to 1ng/ul if possible. For samples with DNA too low to read on broad range Qubit, they were not diluted.
Sample information and dilutions:
| sample # | sample type | ul DNA | ul diluent |
|---|---|---|---|
| A | Puregene | NA | NA |
| B | Puregene | NA | NA |
| 10 | hirt extraction | 2ul | 144ul 10mM tris |
| 11 | hirt extraction | 2ul | 137ul 10mM tris |
| 12 | hirt extraction | 2ul | 66ul 10mM tris |
| 13 | hirt extraction | 2ul | 53ul 10mM tris |
| 14A | hirt extraction | 3ul | 5.5ul 10mM tris |
| 15A | hirt extraction | NA | NA |
| 14 | fly | 2ul | 36ul DNA hydration solution |
| 15 | fly | 2ul | 34ul DNA hydration solution |
| 17 | fly | 2ul | 16ul DNA hydration solution |
| 46 | fly | 2ul | 33ul DNA hydration solution |
| 47 | fly | 2ul | 8ul DNA hydration solution |
| 62 | fly | 3ul | 5.5ul DNA hydration solution |
| 65 | fly | 2ul | 26ulDNA hydration solution |
More information on the hirt samples can be found here. More information on the fly samples can be found here
- Samples were kept on ice
- Reagents were thawed and kept on ice
- Master mixes for each primer were made and kept on ice until use
- TPI master mix
- 255ul Sso
- 25.5ul TPI F primer
- 25.5ul TPI R primer
- 153ul molecular grade water
- PIF3 master mix
- 255ul Sso
- 25.5ul PIF3 F primer
- 25.5ul PIF3 R primer
- 153ul molecular grade water
- 9ul of primer was added to each well of a qPCR plate
- 1ul of diluted (or not) sample was added to the appropriate well in the plate
- The wells were pipette mixed 10X with a multichannel to mix
- The plate was sealed with the biorad seal
- The plate was spun down for 3 minutes at 4000rpm
- The plate was brought to the qPCR machine and run on the p47 program
Plate layout:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | A puregene | A puregene | A puregene | B puregene | B puregene | B puregene | 10 hirt | 10 hirt | 10 hirt | 11 hirt | 11 hirt | 11 hirt | |
| B | 12 hirt | 12 hirt | 12 hirt | 13 hirt | 13 hirt | 13 hirt | 14A hirt | 14A hirt | 14A hirt | 15A hirt | 15A hirt | 15A hirt | TPI |
| C | 14 fly | 14 fly | 14 fly | 15 fly | 15 fly | 15 fly | 17 fly | 17 fly | 17 fly | 46 fly | 46 fly | 46 fly | |
| D | 47 fly | 47 fly | 47 fly | 62 fly | 62 fly | 62 fly | 65 fly | 65 fly | 65 fly | water | water | water | |
| E | A puregene | A puregene | A puregene | B puregene | B puregene | B puregene | 10 hirt | 10 hirt | 10 hirt | 11 hirt | 11 hirt | 11 hirt | |
| F | 12 hirt | 12 hirt | 12 hirt | 13 hirt | 13 hirt | 13 hirt | 14A hirt | 14A hirt | 14A hirt | 15A hirt | 15A hirt | 15A hirt | PIF3 |
| G | 14 fly | 14 fly | 14 fly | 15 fly | 15 fly | 15 fly | 17 fly | 17 fly | 17 fly | 46 fly | 46 fly | 46 fly | |
| H | water | water | water | 47 fly | 47 fly | 47 fly | 62 fly | 62 fly | 62 fly | 65 fly | 65 fly | 65 fly |
All data from the qPCR machine can be found here. Analysis of the hirt samples can be found here. Analysis of the fly samples can be found here.