INTRO
Continuing work with Valentina’s PolyIC project after creating cDNA on 20251019 (Notebook entry), it was time to run the qPCRs.
This notebook describes how the qPCRs were run and links to the various output files. It also provides a brief overview of each primer set’s amplification profiles. This notebook does not have any analysis. This will be performed later.
MATERIALS & METHODS
The following primer sets were run:
| SR ID | Primer Name |
|---|---|
| 1385 | Cg_ATPsynthase_F |
| 1386 | Cg_ATPsynthase_R |
| 1172 | Cg_GAPDH_205_F |
| 1173 | Cg_GAPDH_355_R |
| 599 | Cg_HSP70_F |
| 598 | Cg_HSP70_R |
| 1532 | Cg_Hsp90_F |
| 1533 | Cg_Hsp90_R |
All samples were run in triplicate, on low-profile, white 96-well plates (USA Scientific) sealed with TemPlate TR Select Optical Film (USA Scientific) in a CFX Connect (Bio-Rad) or CFX96 (Bio-Rad) real-time thermalcycler. All reactions consisted of the following:
| Component | Stock Concentration | Volume (uL) |
|---|---|---|
| cDNA | NA | 1 |
| SsoAdvanced Universal SYBR Green Supermix (BioRad) | 2x | 10 |
| PF | 10uM | 0.5 |
| PR | 10uM | 0.5 |
| H2O | NA | 8 |
| TOTAL | 20 |
Master mixes were distributed across three plates for each primer set and included no template controls (NTCs).
For cycling parameters, plate layouts, etc. see the RESULTS section below.
RESULTS
Summary
Files
report-*.pdf: qPCR Reports. Contains plate layouts, cycling params, amp/melt plots, etc.*Cq_Results.csv: Cycle quantity (Cq) data.*.pcrd: Source qPCR data file. Requires CFX Maestro (Bio-Rad) software to open.
ATP synthase
Plate 01
Cq Data
Report
CFX File
Plate 02
Cq Data
Report
CFX File
Plate 03
Cq Data
Report
CFX File
GAPDH
Plate 01
Cq Data
Report
CFX File
Plate 02
Cq Data
Report
CFX File
Plate 03
Cq Data
Report
CFX File
HSP70
Plate 01
Cq Data
Report
CFX File
Plate 02
Cq Data
Report
CFX File
Plate 03
Cq Data
Report
CFX File
HSP90
Plate 01
Cq Data
Report
CFX File
Plate 02
Cq Data
Report
CFX File
Plate 03
Cq Data
Report
CFX File
Plots
