qPCR – Withering Syndrome qPCR Assay Sample Checks

Ran qPCR on water filter samples isolated on 20120127 in an attempt to find water samples that have a higher copy number than Nate’s existing samples for use in the withering syndrome qPCR assay validation.

Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Standard curve was the p16RK7 NcoI-linearized curve made on 20120730.

Baseline threshold was set to 400 and cycles to analyze was set to 41.


qPCR Data File (CFX96):Sam_2012-10-17 15-49-02_CC009827.pcrd

qPCR Report (PDF):Sam_2012-10-17 15-49-02_CC009827.pdf

Everything looked good. Will use sample TAF SD A2 as the high copy number water sample for the withering syndrome qPCR assay.

qPCR – Water Filter Extactions from 20120126

Performed qPCR with WSN1 primer/probe set on the test water filter extractions from 20120126. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). Standards used were Nate’s old standards (no date on tubes/box), as provided by Lisa. The standards are simply labeled as 2x, 3x, 4x, etc. down to 8x. All reactions were run in duplicate.


qPCR Data File (CFX)

qPCR Report (PDF)

The samples analyzed in this qPCR were from The Abalone Farm (TAF), The Cultured Abalone (TCA), and American Abalone (AmA) all at 0 meters (0M), outfall, or drain.

All samples analyzed produced a signal except the American Abalone 0M samples. The Abalone Farm South Drain (SD) samples came up the earliest at ~30 Cq, which corresponds to ~the 5x standard.

These results confirm that the assay is viable for detection of Withering Syndrome from water filter DNA extractions. Will proceed with extracting and qPCR the remaining filters.

Additionally, the standard curve is almost spot on the same as the last time it was run (see 20111221).

UPDATED 20121024 – Modified data file (and subsequently the qPCR Report) to have a baseline threshold of 400 and cycles to analyze 41 to match existing conditions used for the withering syndrome qPCR assay validation.

UPDATE 20140204 – Updated the Ab Endo spreadsheet when Lisa was doing some data analysis and realized that the data she had was much different than what was expected, based on the copy numbers I had entered into the spreadsheet. Turns out, after I updated the baseline threshold on 20121024, I had neglected to update the data in the Ab Endo spreadsheet. That data has now been entered.