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.

Results:

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.

DNA Extraction – Abalone Water Filters from Summer 2010

Extracted DNA from water filters collected during Summer 2010 from various locations using the DNeasy Kit. Based on extraction methodology tests (see 20111221), filter were each cut into ~13 strips and placed in a 1.5mL snap cap tube containing 400uL of Buffer AL and 50uL of Proteinase K. The volumes of both reagents are double the kit recommendation. Samples were vortexed thoroughly and incubated at 56C O/N. After incubation, 400uL (twice the volume in the Qiagen protocol) of 100% EtOH was added to each tube and vortexed thoroughly. The kit protocol was followed for the remainder of the procedure. Samples were eluted with 100uL of Buffer AE and stored @ 4C. Spreadsheet indicating which samples were extracted is here.

DNA Extraction – Abalone Water Filters from Summer 2010

Extracted DNA from water filters collected during Summer 2010 from various locations using the DNeasy Kit. Based on extraction methodology tests (see 20111221), filter were each cut into ~13 strips and placed in a 1.5mL snap cap tube containing 400uL of Buffer AL and 50uL of Proteinase K. The volumes of both reagents are double the kit recommendation. Samples were vortexed thoroughly and incubated at 56C O/N. After incubation, 400uL (twice the volume in the Qiagen protocol) of 100% EtOH was added to each tube and vortexed thoroughly. The kit protocol was followed for the remainder of the procedure. Samples were eluted with 100uL of Buffer AE and stored @ 4C. Spreadsheet indicating which samples were extracted is here.

DNA Extraction – Abalone Water Filters from Summer 2010

Extracted DNA from water filters collected during Summer 2010 from various locations using the DNeasy Kit. Based on extraction methodology tests (see 20111221), filter were each cut into ~13 strips and placed in a 1.5mL snap cap tube containing 400uL of Buffer AL and 50uL of Proteinase K. The volumes of both reagents are double the kit recommendation. Samples were vortexed thoroughly and incubated at 56C O/N. After incubation, 400uL (twice the volume in the Qiagen protocol) of 100% EtOH was added to each tube and vortexed thoroughly. The kit protocol was followed for the remainder of the procedure. Samples were eluted with 100uL of Buffer AE and stored @ 4C. Spreadsheet indicating which samples were extracted is here.

qPCR – DNA Filter Extractions (from 20111123)

Ran qPCR with WSN1 primer/probe set on filter extracts from 20111123 to get a rough idea of how well the extractions worked. 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.

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

None of the filter extractions produced amplification before cycle 40. And, of those that did, only one of each replicate produced a signal. Will have to discuss data with Lisa to see which samples were expected to be “hot” for withering syndrome, if any.

Have decided that the extraction method may be limiting, due to how tight the filter “fits” into the 1.5mL tubes. It seems like the Proteinase K digestion step may not be able to fully coat the filter due to the tight fit. This is problematic, since we are trying to actually quantify the number of WS bugs on each filters. Will test out various filter modifications using test filters from the basement to find a method that makes me feel more comfortable with the potential success of the extraction efficiency.

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.

DNA Extractions – Water Filters from Summer 2010

Extracted DNA from water filters collected during Summer 2010 from various locations using the DNeasy Kit. Samples were eluted with 100uL of Buffer AE and stored @ 4C. Spreadsheet indicating which samples were extracted is here. Samples were quantified on 20111129 using 1uL of template, in 100uL of PicoGreen solution prepared in Low TE. After a few tries, a “low curve” was required to be used to accurately quantify the DNA in these samples.

Results:

y = 4010.4x + 4324.8

d = 115.35

r = 0.99997

Plate layout and average concentration of samples is here.