Pinto abalone eDNA initial qPCR run

PCR protocol

Adapted from Vadopalas et al. 2006.

320 nM each primer
160 nM probe
30 nm ROX (TaqMan EMM includes ROX)

45 cycles alternated between 95 °C (15 s) and 60 °C (60 s)

Total reaction vol (ul) = 15

Template vol (ul) = 2

Stock Conc (uM) Reaction conc (uM) Reaction vol (ul) # Samples Total vol (ul) + 10%
TaqMan Env Master Mix (EMM) 2X 1X 7.5 96 792
Forward Primer 10 0.32 0.48 96 50.688
Reverse Primer 10 0.32 0.48 96 50.688
TaqMan Probe 10 0.16 0.24 96 25.344
10X Exo IPC Mix 10X 1X 1.5 96 158.4
50X Exo IPC DNA 50X 1X 0.3 96 31.68
H2O 2.5 96 264

 

Plate layout

1 2 3 4 5 6 7 8 9 10 11 12
A H20 H20 H20 C01 C01 C01 C01 C01 C01 C02 C02 C02
B C02 C02 C02 C03 C03 C03 C03 C03 C03 C13 C13 C13
C C13 C13 C13 C18 C18 C18 C18 C18 C18 C05 C05 C05
D C05 C05 C05 C07 C07 C07 C07 C07 C07 C14 C14 C14
E C14 C14 C14 C17 C17 C17 C17 C17 C17 D18 D18 D18
F D18 D18 D18 Blank Blank Blank Blank Blank Blank S1 S1 S1
G S2 S2 S2 S3 S3 S3 S4 S4 S4 S5 S5 S5
H S6 S6 S6 S7 S7 S7 S8 S8 S8 S9 S9 S9

qPCR cycle data – standards

qPCR cycle data – samples

Dilution series for standards
4502603077 copies/ul gBlock stock

COI copies/ul final volume ul ul gBlock stock ul H2O Ct
10000000 1350 2.998265619 1347.001734 not tested
S1 1000000 200 20 180 15.92373
S2 100000 200 20 180 19.73577
S3 10000 200 20 180 23.20895
S4 1000 200 20 180 27.04923
S5 100 200 20 180 30.32475
S6 10 200 20 180 36.4099
S7 1 200 20 180 37.34173
S8 0.1 200 20 180 39.07832
S9 0.01 200 20 180 not detected

 

Abalone tank data 

Cohort Settlement Date Family Tank TaskID Inventory Date Total Animals Growth Rate Per Day Mean SL (mm) weight (g) Tank biomass (g)* Ct Estimated copy number per ul
2019 6/6/19 GR03 X GR11 C01 Inventory_1 10/21/20 61 0.049 27.41 2.308005036 140.7883072 32.5555 27.55492515
2019 6/6/19 OR88 X GR05 C02 Inventory_1 10/21/20 146 0.028 15.57 0.377790818 55.15745946 34.41917 8.236068704
2019 7/3/19 OR65 X GR08 C03 Inventory_1 10/21/20 173 0.025 13.59 0.244472549 42.2937509 32.61198 26.56467302
2019 7/3/19 OR96 X GR08 C13 Inventory_1 10/21/20 339 0.02 10.57 0.109387525 37.08237112 33.04966 20.00466729
2019 9/10/19 OR68 X OR57 C18 Inventory_1 10/21/20 226 0.032 14.9 0.328188907 74.17069295 32.47079 29.10975586
2019 7/3/19 OR67 X GR08 C05 Inventory_1 10/27/20 375 0.015 8.07 0.046123595 17.2963481 32.852 22.73826734
2019 7/3/19 OR96 X GR08 C07 Inventory_1 10/27/20 257 0.024 12.9 0.206925928 53.17996347 33.14849 18.76369285
2019 8/21/19 OR94 X GR12 C14 Inventory_1 10/27/20 186 0.037 18.1 0.611644786 113.7659302 32.7927 23.62902173
2019 9/10/19 OR68 X OR57 C17 Inventory_1 10/27/20 251 0.043 20.02 0.844525704 211.9759518 31.17188 67.54317689
2019 9/10/19 GR14 X OR45 D18 Inventory_1 10/27/20 1004 0.03 14.23 0.28325887 284.3919057 30.90362 80.36666944

*equation from: Zhang, Z., Campbell, A. and Lessard, J., 2007. Modeling northern abalone, Haliotis kamtschatkana, population stock and recruitment in British Columbia. Journal of Shellfish Research, 26(4), pp.1099-1107.

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