Tag Archives: QIAamp Fast DNA Stool Mini Kit

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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DNA Isolation – Black Abalone 2nd Experiment 08:13 Accessions

Isolated DNA from EtOH-preserved black abalone digestive gland tissue from the 2nd black abalone experiment.

There’s some odd background in regards to these samples which I previously described here that might be worth reviewing.

DNA was isolated using the QIAamp Fast DNA Stool Kit (Qiagen). Tissues were weighed and briefly homogenized with a disposable pestle in InhibitEX Buffer. Manufacturer’s protocol was followed. DNA  was eluted in 100μL of Buffer ATE and quantified on the Roberts Lab Qubit3.0 (ThermoFisher) using 1μL with the Qubit dsDNA Broad Range assay.

Results:

Google Sheet: 20160421_DNA_isolation_08:13_subset

 

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DNA Isolation – Black Abalone 2nd Experiment 08:13 Accessions

Oddly, I was unable to find any DNA for the 08:13 samples that should have been previously qPCR’d for RLO.

Instead, I tracked down the EtOH-preserved digestive gland (DG) tissues from when these were initially sampled. The box contained both of the “QPCR” tissue samples, however, many of them had dried out. This fact had already been denoted on the outside of the box and on the tubes.

Finding these samples is a bit strange. It’s odd because if someone had performed qPCR analysis on these 08:13 samples, the DNA should’ve come from either of the two “QPCR” tissue samples; but, looking at the vials, it seems like no tissue has been removed from any of the tubes…

Additionally, despite the fact that the spreadsheet Carolyn provided me with the other day indicating that the 08:13 samples are from the 2nd black abalone experiment, the label on this box indicates that these are from the 1st black abalone experiment… Despite this, I’m fairly certain these are indeed from Experiment 2, as these accession numbers have never been brought up before in any of Lisa’s extensive work on the 1st black abalone experiment.

I extracted DNA using the QIAmp Fast DNA Stool Mini Kit (Qiagen) from the following samples. DNA was eluted with 100μL of Buffer ATE and quantified on the Roberts Lab Qubit3.0 (ThermoFisher) using 1μL.

ACCESSION
08:13-2
08:13-3
08:13-4
08:13-5
08:13-6
08:13-7
08:13-11
08:13-12
08:13-13
08:13-14
08:13-16
08:13-17

Results:

Google Sheet: 20160329_DNA_isolation_08:13_subset

Will run qPCRs (WSN1, RLOv DNA helicase, and XenoCal prophage portal) on these samples tomorrow.

DNA has been stored in an existing box in the full-sized -20C freezer in FSH240 and the label on the box has been updated to include these samples.

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