Tag Archives: Qubit 3.0

DNA Isolation – Geoduck gDNA for Illumina-initiated Sequencing Project

We were previously approached by Cindy Lawley (Illumina Market Development) for possible participation in an Illumina product development project, in which they wanted to have some geoduck tissue and DNA on-hand in case Illumina green-lighted the use of geoduck for testing out the new sequencing platform on non-model organisms. Well, guess what, Illumina has give the green light for sequencing our geoduck! However, they need at least 4μg of gDNA, so I’m isolating more.

Isolated DNA from ctenidia tissue from the same Panopea generosa individual used for the BGI sequencing efforts. Tissue was collected by Brent & Steven on 20150811.

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from five separate ~60mg pieces of ctenidia tissue according to the manufacturer’s protocol, with the following changes:

  • Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
  • Incubated homogenate at 60C for 1hr
  • No optional steps were used
  • Performed three rounds of 24:1 chloroform:IAA treatment
  • Eluted each in 50μL of Elution Buffer and pooled into a single sample

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 162ng/μL (Quant data is here [Google Sheet]: 20170105_gDNA_geoduck_qubit_quant

Yield is great (total = ~32μg).

Evaluated gDNA quality (i.e. integrity) by running 162ng (1μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O’GeneRuler DNA Ladder Mix (ThermoFisher).

 

Results:

 

 

DNA looks good: bright high molecular weight band, minimal smearing, and minimal RNA carryover (seen as more intense “smear” at ~500bp).

Will send off 10μg (they only requested 4μg) so that they have extra to work with in case they come across any issues.

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DNA Isolation – Geoduck gDNA for Potential Illumina-initiated Sequencing Project

We were approached by Cindy Lawley (Illumina Market Development) yesterday to see if we’d be able to participate in some product development. We agreed and need some geoduck DNA to send them, in case she’s able to get our species greenlighted for use.

Isolated DNA from ctenidia tissue from the same Panopea generosa individual used for the BGI sequencing efforts. Tissue was collected by Brent & Steven on 20150811.

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from two separate 50mg pieces of ctenidia tissue according to the manufacturer’s protocol, with the following changes:

  • Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
  • Incubated homogenate at 60C for 1hr
  • No optional steps were used
  • Performed three rounds of 24:1 chloroform:IAA treatment
  • Eluted each in 50μL of Elution Buffer and pooled into a single sample

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 19.4ng/μL (Quant data is here [Google Sheet]: 20161221_gDNA_qubit_quant

Yield is low (~1.8μg), but have enough to satisfy the minimum of 1μg requested by Cindy Lawley.

Evaluated gDNA quality (i.e. integrity) by running ~250ng (12.5μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O’GeneRuler DNA Ladder Mix (ThermoFisher).

 

Results:

 

 

 

 

Overall, the sample looks good. Strong, high molecular weight band is present with minimal smearing. However, there is a smear in the ~500bp range. This is most likely residual RNA. This is surprsing since the E.Z.N.A Mollusc Kit includes n RNase step. Regardless, having intact, high molecular weight DNA is the important part for this project. Will prepare to send remainder (~1.5μg) of geoduck to Illumina with other requested samples.

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DNA Isolation – Ostrea lurida DNA for PacBio Sequencing

In an attempt to improve upon the partial genome assembly we received from BGI, we will be sending DNA to the UW PacBio core facility for additional sequencing.

Isolated DNA from mantle tissue from the same Ostrea lurida individual used for the BGI sequencing efforts. Tissue was collected by Brent & Steven on 20150812.

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from two separate 50mg pieces of mantle tissue according to the manufacturer’s protocol, with the following changes:

  • Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
  • Incubated homogenate at 60C for 1.5hrs
  • No optional steps were used
  • Performed three rounds of 24:1 chloroform:IAA treatment
  • Eluted each in 50μL of Elution Buffer and pooled into a single sample

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 326ng/μL (Quant data is here [Google Sheet]: 20161214_gDNA_Olurida_qubit_quant

Yield is good and we have more than enough (~5μg is required for sequencing) to proceed with sequencing.

Evaluated gDNA quality (i.e. integrity) by running ~500ng (1.5μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O’GeneRuler DNA Ladder Mix (ThermoFisher).

Results:

 

 

Overall, the gel looks OK. A fair amount of smearing, but a strong, high molecular weight band is present. The intensity of the smearing is likely due to the fact that the gel is overloaded for this particular well size. If I had used a broader comb and/or loaded less DNA, the band would be more defined and the smearing would be less prominent.

Will submit sample to the UW PacBio facility tomorrow!

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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 & Quantification- Ava Withering Syndrome Transmission Study Water Filters

DNA was extracted from filters using the Qiagen DNeasy Blood & Tissue Kit (spin column protocol). Filters were incubated in 400uL of Buffer AL (twice the volume in the Qiagen protocol in order to completely coat the filters) and 50uL of Proteinase K (twice the volume in the Qiagen protocol) at 56C O/N. After incubation, 400uL (twice the volume in the Qiagen protocol) of 100% EtOH was added to each tube and vortexed thoroughly. The supernatant was transferred to the Qiagen spin columns and the Qiagen protocol was followed. Samples were eluted with 100uL of Buffer AE.

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

The list of samples are listed below.

Results:

Concentrations are very low for both samples. This may, or may not, be expected, depending on volume of water filtered, where it was collected from, etc.

Raw Qubit Readout (Google Sheet): 20160818_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 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 Quantification – Ava Withering Syndrome Transmission Study Samples

DNA samples from yesterday and this morning were quantified using the Roberts Lab Qubit 3.0 (Life Technologies) using the Qubit ds DNA BR reagents. Used 1μL of each sample.

Results:

Raw Qubit readout (Google Sheet): 20160810_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 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 Quantification – Coral DNA from Jose M. Eirin-Lopez (Florida International University)

Quantified the DNA we received from Jose on 20160615 using the Qubit 3.0 Flouorometer (ThermoFisher) with the dsDNA Broad Range (BR) Kit according to the manufacturer’s protocol. Used 1μL of each sample.

Results are here (Google Sheet): Coral_DNA_QubitData_2016-06-30_08-45-56.xls

Here is a table of sample concentrations:

Sample Concentration(ng/μL)
H1 1 52.4
H1 5 34
H1 6 13
H1 8 22
H1 10 39
H1 12 52.4
H5 1 14.7
H5 5 20.8
H5 6 54
H5 8 18.4
H5 10 46.6
H5 12 29.8
H24 1 16.2
H24 5 25
H24 6 20.2
H24 8 22
H24 10 22
H24 12 30.6

 

Will proceed with DNA methylation assessment.

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