Tag Archives: RM B

qPCR – RLOv DNA helicase and XenoCal prophage on Ab Endo Water Filters

Stan Langevin was interested in seeing if the RLOv (phage) and/or the prophage portal genes were detectable in water samples from Lisa’s Ab Endo project.

Ran qPCR on the following samples that Lisa selected:

DNA from water filters collected in 2010. DNA isolated 20120111:

  • CP 0M A
  • CP 0M B
  • MA 0M A
  • MA 0M B
  • PSN 0M A
  • PSN 0M B
  • RM A
  • RM B

DNA from water filters collected in 2011. DNA isolated 20140822:

  • AM Drain 2B
  • PCI SRI PC 1B

RLOv_DNA_helicase master mix calcs are here (Google Sheet): 20161213 – qPCR RLOv DNA Helicase

XenoCal prophage master mix calcs are here (Google Sheet): 20161213 – qPCR XenoCal phage portal

RLOv_DNA_helicase standard curve from 20151224.

All samples were run in duplicate. Plate layout, cycling params, etc. can be seen in the qPCR Report below.

Results:

RLOv_DNA_helicase
qPCR Report (PDF): Sam_2016-12-13 14-52-05_CC009827_RLOv_helicase.pdf
qPCR Data File (CFX): Sam_2016-12-13 14-52-05_CC009827_RLOv_helicase.pcrd

 

XenoCal prophage
qPCR Report (PDF): Sam_2016-12-13 14-52-05_CC009827_XCprophage.pdf
qPCR Data File (CFX): Sam_2016-12-13 14-52-05_CC009827_XCprophage.pcrd

 

  • RLOv DNA helicase amplified in all samples EXCEPT the two samples from 2011. These two samples were negative for the RLO (see Ab Endo sheet “water 2011″).
  •  XC prophage amplfied inconsistently (i.e. replicates did not match/amplify) in only three samples. Additionally, the melt curve of one of those samples differs from the other two. Based on the inconsistencies in technical reps, I should probably repeat this, but technical reps across all of the RLOv DNA helicase samples are very tight, suggesting that my technique was fine (it would be odd if my technique faltered only on ALL of the XC prophage samples)…

 

RLOv DNA HELICASE

 


 

XENOCAL PROPHAGE

 

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qPCR – RLOv DNA Helicase 2010 Water Filter DNA

Since we have a working qPCR for detecting the withering syndrome bacteriophage (RLOv), Carolyn wanted to see how detection/quantification compared to withering syndrome detection/quantification on water samples collected from various farms and the nearest wild abalone site.

DNA samples used were extractions from water filters collected for the Ab Endo Project in 2010.

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20151228 – qPCR RLOv 2010 H2O Filters

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

qPCR LABEL FULL DESCRIPTION FARM/WILD SITE NEAREST FARM/WILD SITE
painted-SCI_B Painted Cave SCI B Wild The Cultured Abalone
painted-SCI_A Painted Cave SCI A Wild The Cultured Abalone
prison-SCI_B Prisoner’s SCI B Wild The Cultured Abalone
prison-SCI_A Prisoner’s SCI A Wild The Cultured Abalone
TCA_out_East_A The Cultured Abalone Outfall East A Farm Santa Cruz Islands
TCA_out_East_B The Cultured Abalone Outfall East B Farm Santa Cruz Islands
TCA_out_West_A The Cultured Abalone Outfall West A Farm Santa Cruz Islands
TCA_out_West_B The Cultured Abalone Outfall West B Farm Santa Cruz Islands
TAF_ND_A1 The Abalone Farm North Drain A1 Farm Pt. Sierra Nevada/Rancho Marina
TAF_SD_A1 The Abalone Farm South Drain A1 Farm Pt. Sierra Nevada/Rancho Marina
TAF_SD_A2 The Abalone Farm South Drain A2 Farm Pt. Sierra Nevada/Rancho Marina
RM_A Rancho Marina A Wild The Abalone Farm
RM_B Rancho Marina B Wild The Abalone Farm
AmA_Drain_A1 American Abalone Drain A1 Farm Carmel
AmA_Drain_A2 American Abalone Drain A2 Farm Carmel
AmA_Drain_B1 American Abalone Drain B1 Farm Carmel
AmA_Drain_B2 American Abalone Drain B2 Farm Carmel
PSN_0M_A Pt. Sierra Nevada 0M A Wild The Abalone Farm
PSN_0M_B Pt. Sierra Nevada 0M B Wild The Abalone Farm
CP_0M_A1 Carmel 0M A1 Wild American Abalone
CP_0M_B Carmel 0M B Wild American Abalone
CP_0M_A Carmel 0M A Wild American Abalone

Results:
qPCR Report (PDF): Sam_2015-12-28 11-20-16_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-12-28 11-20-16_CC009827.pcrd

Overall, data looks good. Will enter copy numbers into the Ab Endo master sheet for later analysis (Google Sheet): Ab Endo Samples

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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.

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