Tag Archives: abalone

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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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

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DNA Extraction & Quantification – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 35 tissue samples provided by Ava. Presumably, the tissues were digestive gland and I believe they were preserved in ethanol. The list of samples are listed below.

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

After extraction, the samples were quantified using the Roberts Lab Qubit 3.0 (Life Technologies) using the Qubit ds DNA BR reagents. Used 1μL of each sample.

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.

Results:

Raw Qubit Readout (Google Sheet): 20160817_DNA_quant_Qubit_Ava_abalone_WS

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 Tissues

Isolated DNA from 30 tissue samples provided by Ava. Presumably, the tissues were digestive gland and I believe they were preserved in ethanol. The list of samples are listed below.

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 will be quantified later today.

Samples were stored at 4C in FSH240 in the boxes “Ava WS Transmission DNA Extractions by Sam Box 1 & 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

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DNA Extraction– Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 20 tissue samples provided by Ava. Presumably, the tissues were digestive gland and I believe they were preserved in ethanol. The list of samples are listed below.

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 will be quantified tomorrow.

Samples were stored at 4C in FSH240 in the box “Ava WS Transmission DNA Extractions by Sam Box 1″. See the master spreadsheet at the bottom of this post for specific sample locations within this box.

 

Cage Accession Weight (mg)
Control 15:09-1 116
Control 15:09-2 68
Control 15:09-3 138
Control 15:09-10 135
Control 15:09-11 96
Control 15:09-22 100
Control 15:09-23 117
Control 15:09-32 51
Control 15:09-33 115
Control 15:09-34 190
Control 15:10-30 103
Control 15:10-31 83
Control 15:10-32 116
Control 15:10-65 190
Control 15:10-66 112
Control 15:11-142 89
Control 15:11-143 94
Control 15:11-144 94
Control 15:11-153 206
Control 15:11-154 116

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

 

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DNA Extraction & Quantification – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 27 tissue samples provided by Ava. Presumably, the tissues were digestive gland and I believe they were preserved in ethanol. The list of samples can be seen in the results below.

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

After extraction, the samples were quantified using the Roberts Lab Qubit 3.0 (Life Technologies) using the Qubit ds DNA BR reagents. Used 1μL of each sample.

Samples were stored at 4C in FSH240 in the box “Ava WS Transmission DNA Extractions by Sam Box 1″. See the master spreadsheet at the bottom of this post for specific sample locations within this box.

Results:

cage_number accession_number concentration(ng/μL)
9 15:10-29 92
2 15:10-40 114
2 15:10-41 102
2 15:10-42 96
2 15:10-43 101
2 15:10-44 128
11 15:8-95 73
11 15:8-96 74
11 15:8-97 73
11 15:8-98 130
11 15:8-99 42
1 15:8-100 106
1 15:8-101 96
1 15:8-102 91
1 15:8-103 79
1 15:8-104 48
1 15:8-105 197
1 15:10-1 43
1 15:10-2 187
1 15:10-3 123
1 15:10-4 83
1 15:10-5 123
27 15:11-30 82
27 15:11-31 121
27 15:11-32 83
27 15:11-33 113
27 15:11-34 66

Raw Qubit readout (Google Sheet): 20160725_DNA_quant_Qubit_Ava_abalone_WS

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

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DNA Extraction & Quantification – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 24 tissue samples provided by Ava. Presumably, the tissues were digestive gland and I believe they were preserved in ethanol. The list of samples can be seen in the results below.

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

After extraction, the samples were quantified using the Roberts Lab Qubit 3.0 (Life Technologies) using the Qubit ds DNA BR reagents. Used 1μL of each sample.

Samples were stored at 4C in FSH240 in the box “Ava WS Transmission DNA Extractions by Sam Box 1″. See the master spreadsheet at the bottom of this post for specific sample locations within this box.

Results:

cage_number accession_number concentration(ng/uL)
21 15:11-89 168
21 15:11-90 69.2
21 15:11-91 70.4
21 15:11-92 58.8
21 15:11-93 61.6
17 15:11-84 48
17 15:11-85 80
17 15:11-86 138
17 15:11-87 68
17 15:11-88 18.2
23 15:9-144 60
23 15:9-145 72
23 15:9-146 121
23 15:9-147 159
23 15:9-148 41.8
20 15:11-100 29
20 15:11-101 133
20 15:11-102 116
20 15:11-103 163
20 15:11-104 162
9 15:10-25 226
9 15:10-26 133
9 15:10-27 182
9 15:10-28 194

 

Raw Qubit readout (Google Sheet): 20160721_DNA_quant_Qubit_Ava_abalone_WS

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

 

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Abalone Sampling – Post-esophagus & Digestive Gland

Helped Ava sample 74 red abalone (Haliotis rufescens).

Accession numbers 15:10-1 through 15:10-74

  • Animals were weighed.

  • Took one post-esophagus sample and one digestive gland sample for histology, placed in histology cassette; three animals per cassette.

  • Took one post-esophagus sample in 95% ethanol for qPCR

  • Shells were labeled and retained for post-sampling weighing/measurements.

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Abalone Sampling – Post-esophageal & Digestive Gland Tissues

Ava sent up abalone for sampling from California for tissue sampling. Here’s the summary of how things went.

  • Animal temps @ -8.0C when opened at lab
  •  ~120 animals sampled
  •  Only 1 dead animal found- Additional dead abalone included in shipment were NOT sampled; too dead
  • Started @ ~11AM, finished at 5:15PM (3 people dissecting, 1 person weighing)
  • All mesh bags were saved and are in a 10% bleach solution over night in sink in 236
  • Histo cassettes put in Davidson’s over night; need to be transferred to 70% EtOH tomorrow
  • Histo cassette layout: animal #1 upper left, animal #2 upper right, animal #3 lower left
  • Tubes for 15:9 sampling have labels applied
  • Histo cassettes for 15:9 sampling have NOT been labelled
  • Empty shells have been labelled and saved; will need to be weighed/measured later

 

 

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qPCR – 2011 Ab Endo Water Filters

Master mix calcs are here: 20150622 – qPCR WS Ab Endo H2O Filters

All samples were run in duplicate. See the qPCR Report (in Results below) for plate layout, cycling params, etc.

Standard curve was p18RK7 from 20120731.

Baseline threshold set to 580, based on calculations by Lisa.

Results:

qPCR Report (PDF): Sam_2015-06-22 14-25-45_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-06-22 14-25-45_CC009827.pcrd

All data was entered in to the Ab Endo Samples spreadsheet.

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