Ran qPCR on the Taylor water filter DNA extracts from 20120322 using V.tubiashii VtpA primers (provide by Elene; no SR ID?) instead of 16s primers, which failed to produce acceptable results in the melt curves (see 20120323). Additionally, Elene has a standard curve for V. tubiashii (from 1/12/2011) based off of CFUs/mL, which will allow us to quantify theoretical number of V.tubiashii CFUs present in each sample.
Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).
qPCR Date File (CFX96)
qPCR Report (PDF)
Overall, the run looks excellent. Both negative controls and no template controls are clean. Since I was able to use a standard curve, I determined CFUs of V.tubiashii in each sample, as follows:
Mean CFUs per qPCR reaction / template volume per qPCR reaction x filter extraction elution volume (100uL) = total CFUs on water filter.
Total CFUs on filter / filtered water volume = CFUs per mL in Taylor tanks
158 - 16500 copies/2uL = 8250 copies/uL x 100uL = 825000 copies on water filter/1000mL = 825 copies/mL
200 - 5700 copies/2uL = 2850 copies/uL x 100uL = 285000 copies on water filter/1000mL = 285 copies/mL
279 - 325000 copies/2uL = 162500 copies/uL x 100uL = 16250000 copies on water filter/1000mL = 16250 copies/mL
313 - 152 copies/2uL = 76 copies/uL x 100uL = 7600 copies on water filter/1000mL = 7.6 copies/mL
341 - 124000/2uL = 62000 copies/uL x 100uL = 6200000 copies on water filter/1000mL = 6200 copies/mL
410 - 132000/2uL = 66000 copies/uL x 100uL = 6600000 copies on water filter/1000mL = 6600 copies/mL
433 - 63700/2uL = 31850 copies/uL x 100uL = 3185000 copies on water filter/1000mL = 3185 copies/mL
503 - 110/2uL = 55 copies/uL x 100uL = 5500 copies on water filter/1000mL = 5.5 copies/mL
551 - 2000/2uL = 1000 copies/uL x 100uL = 100000 copies on water filter/1000mL = 100 copies/mL
604 - 272/2uL = 136 copies/uL x 100uL = 13600 copies on water filter/1000mL = 13.6 copies/mL
Sample #410 was from the only tank that exhibited mortalities and was the only group of oyster larvae that showed any expression from the V.tubiashii genes (see DATE).