I ran remaining field samples over the past 3 days or so. qPCR conditions were identical to those reported earlier except that cycle count was increased to 50 (plate 4) and 55 (plate 5). Plate configurations and results were as follows:
| Plate 4 | 3/27/21 | |||||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | H20 | H20 | H20 | S2 | S2 | S2 | Swi0m | Swi0m | Swi0m | Swi0m | Swi0m | Swi0m |
| B | S3 | S3 | S3 | S4 | S4 | S4 | Ava-S | Ava-S | Ava-S | Ava-S | Ava-S | Ava-S |
| C | S5 | S5 | S5 | S6 | S6 | S6 | Ava2m | Ava2m | Ava2m | Ava2m | Ava2m | Ava2m |
| D | S7 | S7 | S7 | S8 | S8 | S8 | Ava0m | Ava0m | Ava0m | Ava0m | Ava0m | Ava0m |
| E | Gol2m | Gol2m | Gol2m | Gol2m | Gol2m | Gol2m | RS311 | RS311 | RS311 | RS311 | RS311 | RS311 |
| F | Gol-S | Gol-S | Gol-S | Gol-S | Gol-S | Gol-S | Hus-S | Hus-S | Hus-S | Hus-S | Hus-S | Hus-S |
| G | Swi-S | Swi-S | Swi-S | Swi-S | Swi-S | Swi-S | Hus2m | Hus2m | Hus2m | Hus2m | Hus2m | Hus2m |
| H | Swi2m | Swi2m | Swi2m | Swi2m | Swi2m | Swi2m | Hus0m | Hus0m | Hus0m | Hus0m | Hus0m | Hus0m |
| Plate 5 | 3/29/21 | |||||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | H20 | H20 | H20 | S2 | S2 | S2 | Sm210 | Sm210 | Sm210 | Sm210 | Sm210 | Sm210 |
| B | S3 | S3 | S3 | S4 | S4 | S4 | Inf-S | Inf-S | Inf-S | Inf-S | Inf-S | Inf-S |
| C | S5 | S5 | S5 | S6 | S6 | S6 | Inf2m | Inf2m | Inf2m | Inf2m | Inf2m | Inf2m |
| D | S7 | S7 | S7 | S8 | S8 | S8 | Inf0m | Inf0m | Inf0m | Inf0m | Inf0m | Inf0m |
| E | Sla-S | Sla-S | Sla-S | Sla-S | Sla-S | Sla-S | Dra0m | Dra0m | Dra0m | Dra0m | Dra0m | Dra0m |
| F | Sla2m | Sla2m | Sla2m | Sla2m | Sla2m | Sla2m | Om0m | Om0m | Om0m | Om0m | Om0m | Om0m |
| G | Sla0m | Sla0m | Sla0m | Sla0m | Sla0m | Sla0m | Bay0m | Bay0m | Bay0m | Bay0m | Bay0m | Bay0m |
| H | RS029 | RS029 | RS029 | RS029 | RS029 | RS029 | Ava0m | Ava0m | Ava0m | Ava0m | Ava0m | Ava0m |
To summarize, the only field site where eDNA was detected in these two qPCR runs was a single replicate of Baytown 0m (Bay0m). This is notable because this is one of the four sites/samples that were re-run. The previous run did not detect any eDNA in Baytown 0m. This is likely to be simply a chance amplification of eDNA occurring at low abundance in the DNA extract; in other words, with enough replicates, many of these sites would probably show positive results. This argues for measures such as (1) increasing water filtrate volumes, (2) concentrating DNA extracts, and/or (3) increasing template concentrations in the qPCR. I think options 1 and 3 make the most sense and suggest increasing filtrate volumes from 1L to 2L and perhaps doubling qPCR reaction volumes from 15 μl to 30 μl.