Tag Archives: DNA Isolation

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 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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DNA Isolation – Oly gDNA for BS-seq

Need DNA to prep our own libraries for bisulfite-treated high-throughput sequencing (BS-seq).

Isolated gDNA from the following tissue samples stored in RNAlater (tissue was not weighed) using DNAzol:

2NF1
2NF2
2NF3
2NF4
2NF5
2NF6
2NF7
2NF8
1NF11
1NF12
1NF13
1NF14
1NF15
1NF16
1NF17
1NF18

The sample coding breaks down as follows (see the project wiki for a full explanation):

2NF#

2 = Oysters outplanted in Fidalgo Bay

NF = Broodstock originated in Fidalgo Bay

= Sample number

1NF#

1 = Oysters outplanted in Oyster Bay

NF = Broodstock originated in Fidalgo Bay

= Sample number

 

DNA was isolated in the following manner:

  • Homogenized tissues in 500μL of DNAzol (Molecular Research Center; MRC).
  • Added additional 500μL of DNAzol.
  • Added 10μL of RNase A (10mg/mL, ThermoFisher); incubated 10mins @ RT.
  • Added 300μL of chloroform and mixed moderately fast by hand.
  • Incubated 5mins @ RT.
  • Centrifuged 12,000g, 10mins, RT.
  • Transferred aqueous phase to clean tube.
  • Added 500μL of 100% EtOH and mixed by inversion.
  • Pelleted DNA 5,000g, 5mins @ RT.
  • Performed 3 washes w/70% EtOH.
  • Dried pellet 3mins.
  • Resuspended in 100μL of Buffer EB (Qiagen).
  • Centrifuged 12,000g, 10mins, RT to pellet insoluble material.
  • Transferred supe to clean tube.

The samples were quantified using the Qubit dsDNA BR reagents (Invitrogen) according to the manufacturer’s protocol and used 1μL of sample for measurement.

Results:

Qubit data (Google Sheet): 20151216_Oly_gDNA_qubit_quants

SAMPLE CONCENTRATION (ng/μL)
2NF1 76.4
2NF2 175
2NF3 690
2NF4 11.7
2NF5 142
2NF6 244
2NF7 25
2NF8 456
1NF11 182
1NF12 432
1NF13 155
1NF14 21
1NF15 244
1NF16 112
1NF17 25.2
1NF18 278

 

Will run samples on gel tomorrow to evaluate gDNA integrity.

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DNA Isolation – Olympia Oyster Outer Mantle gDNA

Isolated additional gDNA for the genome sequencing. To try to improve the quality (260/280 & 260/230 ratios) of the gDNA, I added a chloroform step after the initial tissue homogenization.

Used 123mg of Ostrea lurida outer mantle collected by Brent & Steven on 20150812.

  • Homogenized in 500μL of DNAzol.
  • Added additional 500μL of DNAzol.
  • Centrifuged 12,000g, 10mins, @ RT.
  • Split supernatant equally into two tubes.
  • Added 500μL of chloroform and mixed moderately fast by hand.
  • Centrifuged 12,000g, 10mins, RT.
  • Combined aqueous phases from both tubes in a clean tube.
  • Added 500μL of 100% EtOH and mixed by inversion.
  • Spooled precipitated gDNA and transferred to clean tube.
  • Performed 3 washes w/70% EtOH.
  • Dried pellet 3mins.
  • Resuspended in 200μL of Buffer EB (Qiagen).
  • Centrifuged 10,000g, 5mins, RT to pellet insoluble material.
  • Transferred supe to clean tube.

DNA was quantified using two methods: NanoDrop1000 & Qubit 3.0 (ThermoFisher).

For the Qubit, the samples were quantified using the Qubit dsDNA BR reagents (Invitrogen) according to the manufacturer’s protocol and used 1μL of sample for measurement.

Results:

Qubit Data (Google Sheet): 20151125_qubit_gDNA_geoduck_oly_quants

METHOD CONCENTRATION (ng/μL) TOTAL (μg)
Qubit 137 27.4
NanoDrop1000 295 59.0

 

Yield is solid. We should finally have sufficient quantities of gDNA to allow for BGI to proceed with the rest of the genome sequencing! Will run sample on gel to evaluate integrity and then send off to BGI.

The NanoDrop & Qubit numbers still aren’t close (as expected).

The addition of the chloroform step definitely helped improve the 260/280 OD ratio (see below). However, the addition of that step had no noticeable impact on the 260/230 OD ratios, which is a bit disappointing.

 

NanoDrop Absorbance Values & Plots

 

 

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DNA Isolation – Geoduck Ctenidia gDNA

Isolated additional gDNA for the genome sequencing. In an attempt to obtain better yields, I used ctenidia (instead of adductor muscle). Additionally, to try to improve the quality (260/280 & 260/230 ratios) of the gDNA, I added a chloroform step after the initial tissue homogenization.

Used 190mg of Panopea generosa ctenidia collected by Brent & Steven on 20150811.

  • Homogenized in 500μL of DNAzol.
  • Added additional 500μL of DNAzol.
  • Centrifuged 12,000g, 10mins, @ RT.
  • Split supernatant equally into two tubes.
  • Added 500μL of chloroform and mixed moderately fast by hand.
  • Centrifuged 12,000g, 10mins, RT.
  • Combined aqueous phases from both tubes in a clean tube.
  • Added 500μL of 100% EtOH and mixed by inversion.
  • Spooled precipitated gDNA and transferred to clean tube.
  • Performed 3 washes w/70% EtOH.
  • Dried pellet 3mins.
  • Resuspended in 200μL of Buffer EB (Qiagen).
  • Centrifuged 10,000g, 5mins, RT to pellet insoluble material.
  • Transferred supe to clean tube.

DNA was quantified using two methods: NanoDrop1000 & Qubit 3.0 (ThermoFisher).

For the Qubit, the samples were quantified using the Qubit dsDNA BR reagents (Invitrogen) according to the manufacturer’s protocol and used 1μL of sample for measurement.

Results:

Qubit Data (Google Sheet): 20151125_qubit_gDNA_geoduck_oly_quants

METHOD CONCENTRATION (ng/μL) TOTAL (μg)
Qubit 105 21.0
NanoDrop1000 173 34.6

 

Yield is definitely much, much better than adductor muscle! Should’ve switched to a different tissue a long time ago! We should finally have sufficient quantities of gDNA to allow for BGI to proceed with the rest of the genome sequencing! Will run sample on gel to evaluate integrity and then send off to BGI.

The NanoDrop & Qubit numbers still aren’t close (as expected).

The addition of the chloroform step definitely helped improve the 260/280 OD ratio (see below). However, the addition of that step had no noticeable impact on the 260/230 OD ratios, which is a bit disappointing.

 

NanoDrop Absorbance Values & Plots

 

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DNA Isolation – Geoduck Adductor Muscle gDNA

Since we still don’t have sufficient gDNA for the full scope of the genome sequencing, I isolated more gDNA.

Isolated gDNA from 257mg adductor muscle tissue collected by Steven & Brent on 20150811.

Tissue was thoroughly minced with a clean razor blade and then processed with the E.Z.N.A. Mollusc Kit (Omega BioTek) with the following changes:

  • Doubled solution volumes for steps before sample was loaded on columns
  • Sample was split equally in two tubes prior to addition of 100% EtOH
  • All mixing was done by shaking – no vortexing! Done this way to, hopefully, maintain gDNA integrity
  • Elution volume = 50μL
  • Elution was repeated using the initial elution to maximize recovery while maintaining low sample volume.
  • The two preps were pooled – final volume = 79μL

DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit

For the Quant-IT kit, the samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

METHOD CONCENTRATION (ng/μL) VOLUME (μL) YIELD (ng)
NanoDrop1000 54.93 79 4,339
Quant-IT 34.52 79 2,727

 

The NanoDrop1000 overestimates the concentration of the sample by 1.6x!

Regardless, the yield isn’t all that great, which has generally been the case for all of the geoduck gDNA isolations I’ve performed. It would probably be prudent to try isolating gDNA from a different tissue to see if yields improve…

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants

 

NanoDrop1000 Measurements and Plots

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DNA Isolation – Olympia Oyster Outer Mantle gDNA

Since we still don’t have sufficient gDNA for the full scope of the Olympia oyster genome sequencing, I isolated more gDNA.

Isolated gDNA from 118mg outer mantle tissue collected by Steven & Brent on 20150812.

Tissue was thoroughly minced with a clean razor blade and then processed with the E.Z.N.A. Mollusc Kit (Omega BioTek) with the following changes:

  • Doubled solution volumes for steps before sample was loaded on columns
  • Sample was split equally in two tubes prior to addition of 100% EtOH
  • All mixing was done by shaking – no vortexing! Done this way to, hopefully, maintain gDNA integrity
  • Elution volume = 50μL
  • Elution was repeated using the initial elution to maximize recovery while maintaining low sample volume.
  • The two preps were pooled – final volume = 79μL

DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit

For the Quant-IT kit, the samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

METHOD CONCENTRATION (ng/μL) VOLUME (μL) YIELD (ng)
NanoDrop1000 552.53 79 43,650
Quant-IT 219.07 79 17,307

 

The NanoDrop1000 overestimates the concentration of the sample by 2.5x!

Regardless, this is a solid yield and, when combined with the other Ostrea lurida gDNA that I cleaned up today, should push the total amount of gDNA submitted to BGI over the required threshold.

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants

 

NanoDrop1000 Measurements and Plots

 

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DNA Isolations – Oly Fidalgo 2SN Ctenidia

Isolated DNA from 24 2SN ctenidia samples from Friday’s sampling (#32 – 55). Samples were thawed at RT.

DNA was isolated using the E.Z.N.A. Mollusc Kit (Omega BioTek) according to the manufacturer’s protocol with the following changes:

  • Samples were incubated @ 60C for only 1hr, per Steven’s recommendation (an attempt to prevent degradation)
  • No optional steps were performed
  • Used 300μL of MBL Buffer for all samples (this was more than the recovered volume of aqueous phase from each sample)
  • Single elution of 50μL

Samples were stored @ -20C in: Oly gDNA Oly Reciprocal Transplant Final Sampling Box #1.

Some notes:

  • Total time (including 1hr incubation): 4.5hrs.
  • Short incubation time did not completely digest samples
  • Partial tissue digestions led to difficulties in recovering entire aqueous phase, post chloroform treatment

 

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DNA Isolation – Geoduck & Olympia Oyster

Amazingly, we need more gDNA for the two genome sequencing projects (geoduck and Olympia oyster). Used geoduck adductor muscle sample from Box 1 of the geoduck samples collected by Brent & Steven on 20150811. Used Olympia oyster ctenidia from Box 1 of adductor muscle sample collected by Brent & Steven on 20150812.

Tissues were split in approximately half, minced and transferred to tubes with 1mL of DNAzol + 50μg/mL of Proteinase K (Fermentas). Previously, I had just homogenized samples. I’m hoping that the overnight digestion with Proteinase K will help increase yields from these.

Tissue weights:

  • Geoduck adductor muscle tube 1: 292mg (gone)
  • Geoduck adductor muscle tube 2: 320mg (gone)
  • Olympia oyster ctenidia tube 1: 135mg (gone)
  • Olympia oyster ctenidia tube 2: 130mg (gone)

Samples were isolated using DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

 

  • Samples were incubated O/N @ RT on a rotator.
  • After Proteinase K digestion, added 40μL RNAse A (100mg/mL) and incubated @ RT for 15mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended Buffer EB (Qiagen)

Resuspension volume = 500μL total for each species

Samples were incubated O/N at RT to facilitate pellet resuspension.

NOTE: Geoduck “pellets” were not very DNA pellet-like. Very loose, white, and sort of disintegrate (but not dissolve in solution) when attempted to resuspend.

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