Category Archives: Ava's Abalone RLO Transmission Experiment

Ava's Abalone RLO Transmission Experiment

qPCR – Ava’s RLO Transmission Samples

Ran qPCRs on samples extracted earlier today. Also re-ran samples 15:08-29 and 15:09-145, per Ava’s request.

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20170619_qPCR_WSN1_Ava_Samples

Plate layouts, cycling params, etc. can be seen in the corresponding qPCR Reports (see Results below).

Baseline threshold was manually set to 580, based on Lisa’s development of the withering syndrome qPCR assay.

Results:

qPCR Report (PDF): Sam_2017-06-19 13-10-22_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-06-19 13-10-22_CC009827.pcrd

 

These will need to be re-run, as the standard curve is a bit wonky (see below). Will re-run later this week.

 

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DNA Extraction – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 19 red abalone digestive gland tissue samples.

Tissue was weighed and then DNA extracted.

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

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was 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 racks for qPCR later today.

Sample information is in this spreadsheet (Google Sheet): Ava WS Transmission DNA Extractions

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qPCR – Ava’s RLO Transmission Samples

Ran qPCRs on the DNA I extracted on 20170504 and earlier today.

The full list of samples is here (Google Sheet): 20170502_Ava_Ab_List

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20170509_qPCR_WSN1_Ava_Samples

Plate layouts, cycling params, etc. can be seen in the corresponding qPCR Reports (see Results below).

Baseline threshold was manually set to 580, based on Lisa’s development of the withering syndrome qPCR assay.

Results:

Curves look good on all runs (except the one that’s been noted and has been repeated). Will pass along to Ava and Carolyn.

qPCR Report (PDF): Sam_2017-05-09 07-29-36_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 07-29-36_CC009827.pcrd

 


This plate has a bad curve and needs to be re-run! It has been repeated below!

I’ve included this for posterity only!

qPCR Report (PDF): Sam_2017-05-09 08-56-22_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 08-56-22_CC009827.pcrd


 

 

qPCR Report (PDF): Sam_2017-05-09 10-21-15_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 10-21-15_CC009827.pcrd

qPCR Report (PDF): Sam_2017-05-09 11-44-42_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 11-44-42_CC009827.pcrd

 

qPCR Report (PDF): Sam_2017-05-09 13-07-46_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 13-07-46_CC009827.pcrd

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DNA Quantification – Ava’s RLO Transmission DNA

Quantified the DNA I isolated on 20170504 and earlier today using the Roberts Lab’s Qubit 3.0 and the dsDNA Broad Range assay.

Used 1uL of each sample.

Results:

The following samples were below the level of sensitivity of the Qubit assay:

  • 15:09-142
  • 15:11-113
  • 15:11-147
  • 15:11-149

Qubit output data (Google Sheet): 20170509_Ava_RLO_quantification_qubit

An easier-to-read summary of all the samples is here (Google Sheet): 20170502_Ava_Ab_List

 

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DNA Extraction – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from five tissue samples.

Tissue was weighed and then DNA extracted.

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

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was 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 racks for qPCR and quantification later today.

Sample information is in this spreadsheet (Google Sheet): 20170502_Ava_Ab_List

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DNA Extraction – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 117 tissue samples that I weighed out yesterday.

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

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was 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 racks for qPCR and quantification next week.

Sample information is in this spreadsheet (Google Sheet): 20170502_Ava_Ab_List

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qPCR – Ava’s RLO Transmission Samples Re-runs

The final plate from my runs of Ava’s samples had a bad standard curve, so I’m re-running them. Additionally, Ava has asked me to add some additional samples. Here are the additional samples:

51
120
135
15:11-86
15:11-30

Sample 120 did not have any volume left (because I used the rest of that sample during the previous qPCR), but, for kicks, I added 5uL of nuclease-free water to it and ran it anyway.

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20170406_qPCR_WSN1_Ava_Samples

Plate layouts, cycling params, etc. can be seen in the corresponding qPCR Reports (see Results below).

Baseline threshold was manually set to 580, based on the Lisa’s development of the withering syndrome qPCR assay.

Results:

qPCR Report (PDF): Sam_2017-04-06 07-53-11_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-04-06 07-53-11_CC009827.pcrd

Curve looked good. Will let Ava know that all the samples are finished.

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qPCR – Ava’s RLO Transmission Samples

Ava provided me with a list of samples that needed to be qPCR’d (Google Sheet): qPCR redos 30117.xlsx

Here’s a list of samples that had no liquid left in them (likely due to evaporation). I added 5uL of nuclease-free water to each sample in hopes of gleaning some data from them:

14
22
37
38
46
48
49
50
52
55
61
65
116
127
149
152
155
157
158

The following samples are samples that I used the remainder of them for these qPCR reactions:

60F1
120
136

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20170322 – qPCR WSN1 Ava Samples 01

Plate layouts, cycling params, etc. can be seen in the corresponding qPCR Reports (see Results below).

Baseline threshold was manually set to 580, based on the Lisa’s development of the withering syndrome qPCR assay.

 

Results:

All but the final plate look good (standard curve-wise). Will re-run last plate next week.

qPCR Report (PDF): Sam_2017-03-22 07-24-02_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 07-24-02_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-22 08-54-50_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 08-54-50_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-22 10-25-58_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 10-25-58_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-22 11-54-57_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 11-54-57_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-22 13-23-37_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 13-23-37_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-22 14-51-55_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 14-51-55_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-22 16-19-59_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 16-19-59_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-23 06-54-02_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-23 06-54-02_CC009827.pcrd

 

NOTE: This needs to be re-run, due to a wonky rep of one of the points of the standard curve.
qPCR Report (PDF): Sam_2017-03-23 08-24-59_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-23 08-24-59_CC009827.pcrd

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DNA Quantification – Ava Withering Syndrome Transmission Study Samples

DNA samples from 20160818 (water filters) and 20160825 (feces) were quantified using the Roberts Lab Qubit 3.0 (Life Technologies) using the Qubit ds DNA HS (high sensitivity) reagents. Used 5μL of each sample.

Results:

Raw Qubit readout (Google Sheets):

 

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

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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

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