DNA Extraction – Ava Withering Syndrome Transmission Study Feces

Isolated DNA from 36 fecal samples provided by Ava. Fecal samples were on filters stored at -80C. Samples were thawed slightly to allow unrolling of the filters. Feces was transferred to pre-weighed tubes and then weighed.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

Samples were stored at 4C in FSH240 in the boxes “Ava WS Transmission DNA Extractions by Sam Box 2″. See the master spreadsheet at the bottom of this post for specific sample locations within this box.

Samples will be quantified tomorrow.

See two pictures below for potential anomalous samples.

This sample had a date of 9/24/15. Possibly incorrect, as no other samples have this date.

 

 

This sample contained no feces at all. Additionally, the filter was a different type than all the other fecal samples. It looks like a water sample filter. Collected liquid from filter and processed that.

 

Master spreadsheet for these, and future, samples for this project (Google Sheet): Ava WS Transmission DNA Extractions

DNA Extraction – Ava Withering Syndrome Transmission Study Feces

Isolated DNA from 4 fecal samples provided by Ava. Fecal samples were on filters stored at -80C. Samples were thawed slightly to allow unrolling of the filters. Feces was transferred to pre-weighed tubes and then weighed.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

Samples were stored at 4C in FSH240 in the boxes “Ava WS Transmission DNA Extractions by Sam Box 2″. See the master spreadsheet at the bottom of this post for specific sample locations within this box.

Master spreadsheet for these, and future, samples for this project (Google Sheet): Ava WS Transmission DNA Extractions

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 thermal cycler.

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 (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.