PCR – Withering Syndrome Convetional PCR Assay Validation (Repeatibility/Reproducibility)

This is an exact repeat of the run from 20121220. Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was the 30,000 copy from the WS standard curve plasmid made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the Eppendorf thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

PCR – Withering Syndrome Convetional PCR Assay Validation (Repeatibility/Reproducibility)

This is an exact repeat of the run from 20121220. Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was the 30,000 copy from the WS standard curve plasmid made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the Eppendorf thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

PCR – Withering Syndrome Convetional PCR Assay Validation (Repeatibility/Reproducibility)

This is an exact repeat of the run from 20121220. Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was the 30,000 copy from the WS standard curve plasmid made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the MJ thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

PCR – Withering Syndrome Convetional PCR Assay Validation (Repeatibility/Reproducibility)

This is an exact repeat of the run from 20121217. Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was the 30,000 copy from the WS standard curve plasmid made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the MJ thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

PCR – Withering Syndrome Conventional PCR Assay Validation (Repeatibility/Reproducibility)

Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was 30,000 copy from the WS standard curve made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the MJ thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

Hyperladder V was used as molecular weight markers.

qPCR – Withering Syndrome qPCR Assay Validation: Reproducibility (CFX96)

Ran additional reps of the Low and Medium Tissue samples, due to poor replication in an earlier run on these samples.

Tissue

  • Low: 09:16-18
  • Med: 09:16-22

Master mix calcs are here.

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

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

qPCR – Withering Syndrome qPCR Assay Validation: Reproducibility (Opticon 2)

Run on the Roberts’ Lab Opticon 2 as part of the reproducibility aspect of assay validation. Master mix calcs are here.

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.

Results:

qPCR Data File (Opticon 2)

qPCR Report (Google Spreadsheet)

Things looked good. Will have Lisa proceed with her part of the reproducibility assay validation.

qPCR – Repeat of Earlier qPCR with New Sample

Re-ran the qPCR (see earlier entry from today), due to the Low Feces samples failing to amplify. Replaced it with:

R3E 5/14/09 – 10^0

Everything else (including master mix, cycling params, etc) is all the same. See the earlier entry for details.

Results:

qPCR Data File (CFX96): Sam_2012-10-22 13-37-42_CC009827.pcrd

qPCR Report (PDF): Sam_2012-10-22 13-37-42_CC009827.pdf

Things looked good. The replacement Low Feces sample amplified.

qPCR – Withering Syndrome qPCR Assay Validation: Reproducibility (CFX 96)

Ran qPCR for the reproducibility aspect of the WSN qPCR Assay Validation. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the Results (see below).

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.

Samples used for “low”, “medium” and “high” copy numbers for each sample type are below, with expected fold copy number (based off of previous qPCRs):

Feces

Low: R4E 4/17/09 – 10^0

Med: R3E 7/23/09 – 10^3

High: R4E 7/23/09 – 10^4

Tissue

Low: 09:16-18 – 10^1

Med: 09:16-22 – 10^2

High: 09:20-11 – 10^5

Water

Low: 494:11-11 – 10^0

Med: 494-11-12 – 10^2

High: TAF SD A2 – 10^3

Results:

qPCR Data File (CFX96): Sam_2012-10-22 16-16-19_CC009827.pcrd

qPCR Report (PDF): Sam_2012-10-22 16-16-19_CC009827.pdf

Everything looked good except for the Low Feces sample which didn’t produce any amplification. Will identify another sample to use for the Low Feces sample.