Testing Amount of DiNV DNA Ratio Between Hirt and Exonuclease Treated DNA With More qPCR
Because the exonuclease treated samples are combined in the cleanup, I have to combine the hirt extracted samples too to make it a fair and paired comparison. Then these are diluted 1:10 to prepare for qPCR.
All samples are handled on ice and flicked and spun down before use.
Pool 1:
- 2ul sample 20
- 2ul sample 21
- 2ul sample 24
- 54ul 10mM tris HCl
Pool 2:
- 2ul sample 22
- 2ul sample 23
- 2ul sample 25
- 54ul 10mM tris HCl
Pool 3:
- 2ul sample 26
- 2ul sample 27
- 2ul sample 28
- 2ul sample 29
- 72ul 10mM tris HCl
Pool 4:
- 3ul sample 30
- 3ul sample 31
- 54ul 10mM tris HCl
Also using the 1:10 diluted samples 20-exo and 22-exo that were already made up previously. But samples 26-exo and 30-exo needed to be diluted 1:10: this was done by combining 3ul of the sample with 27ul of 10Mm Tris HCl
All samples were flick mixed and spun down before use in qPCR
qPCR mixes:
| reagent | TPI | PIF 3 |
|---|---|---|
| Sso | 125ul | 125ul |
| F primer | 12.5ul | 12.5ul |
| R primer | 12.5ul | 12.5ul |
| molec grade water | 75ul | 75ul |
9ul of reagent mix was added to each well in the plate as planned (see layout below). 1ul of DNA was added to each well as planned, and samples were run in triplicate. After addition of DNA each sample well was pipette mixed 10 times, then the plate was sealed and spun down.
The plates was run on the p47 program in the KMM folder.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | primer | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | pool 1 | pool 1 | pool 1 | pool 2 | pool 2 | pool 2 | pool 3 | pool 3 | pool 3 | pool 4 | pool 4 | pool 4 | TPI |
| B | 20-exo | 20-exo | 20-exo | 22-exo | 22-exo | 22-exo | 26-exo | 26-exo | 26-exo | 30-exo | 30-exo | 30-exo | TPI |
| C | pool 1 | pool 1 | pool 1 | pool 2 | pool 2 | pool 2 | pool 3 | pool 3 | pool 3 | pool 4 | pool 4 | pool 4 | PIF 3 |
| D | 20-exo | 20-exo | 20-exo | 22-exo | 22-exo | 22-exo | 26-exo | 26-exo | 26-exo | 30-exo | 30-exo | 30-exo | PIF 3 |
| E | |||||||||||||
| F | |||||||||||||
| G | |||||||||||||
| H |
qPCR data can be found here
Analysis is here, and this is a combined analysis with previous data