Using cDNA from 20230713, performed qPCR on the following primer sets selected by Steven:
| SRID | Primer_name |
|---|---|
| 1409 | Cg_18s_R |
| 1408 | Cg_18s_F |
| 1171 | Cg_Actin_408_R |
| 1170 | Cg_Actin_306_F |
| 1161 | Cg_Def_R |
| 1160 | Cg_Def_F |
| 256 | IL-17 internal RV |
| 255 | IL-17 internal FW |
qPCR calcs:
20230719 - qPCR Calcs Noah gigas ctenidia cDNA (Google Sheet)
- Used same calculations from 20230719.
Reactions were run on white, low-profile, 96-well qPCR plates (USA Scientific). A total of two plates were run. One was run on a CFC Connect (BioRad) and the other on a CFX96 (BioRad). See the qPCR Report in the RESULTS section below for cycling params, plate layouts, etc.
All samples were run in singular. Normally duplicates/triplicates are run. However, due to time and sample constraints for Noah’s project/poster, samples were just run in singular. No template controls were run in duplicate.
RESULTS
qPCR Results file (CSV)
qPCR Data file (QPCRD - CFX Maestro required)
qPCR Report (PDF)
Here’s a brief summary of the qPCR data, based on looking at the amplification and melt curve plots below.
18s: No Template Controls (NTCs) show contamination in melt curve. Since this has shown up previously (notebook), I suspect my primer working stocks have become contaminated. Might not redo, if actin is acceptable for normalization.
actin: Melt curve looks okay - not ideal. Amplification looks good.
defensin: Melt curve looks good. Amplification comes up late, but looks good.
IL-17: Melt curve looks good. Amplification looks good. Sample THM1 might be iffy, as it is an outlier in the 18s primer set as well.
PLOTS
NOTE: NTCs are denoted by red lines in all plots.
| SRID | Primer name | Amplifcation Plot | Melt Curve |
|---|---|---|---|
| 1408/9 | Cg_18s |  |
 |
| 255/6 | Cg_IL17 |  |
 |
| 1170/1 | Cg_actin |  |
 |
| 1160/1 | Cg_defensin |  |
 |