Ethanol Precipitation – Geoduck & Olympia oyster gDNA

Pooled all of the geoduck gDNA from all the previous geoduck isolations for the Geoduck Genome Sequencing Project and pooled all of the Ostrea lurida gDNA from previous Ostrea lurida isolations for the Olympia Oyster Genome Sequencing Project.

Both sets of gDNA were ethanol (EtOH) precipitated. This was done for two reasons – to concentrate the samples to the minimum necessary concentration required by BGI (>119ng/μL) and to try to improve the poor 260/230 ratios (which are likely due to high salt carryover).

Precipitation was performed by consolidating each species of DNA in 15mL conicals, adding 0.1 volumes of 3M sodium acetate (pH= 5.2) and then adding 2.5 volumes of ice cold (-20C) 100% EtOH. Volumes used are below.

Sample DNA Vol (μL) 3M Sodium Acetate Vol. (μL) 100% EtOH Vol (μL) Total Vol (μL)
Geoduck 1860 186 5115 7161
Oly 780 78 2145 3003

Samples were mixed by inversion and incubated @ -80C for 3hrs.

DNA was pelleted by spinning tubes at 12,000g for 30mins @ 4C in a SL-50T (Sorval) rotor in a T21 (Sorval) table top centrifuge.

Supernatant was decanted and pellets were transferred to 1.5mL snap cap tubes.

Pellets were washed three times with 70% EtOH.

After the last wash, the supe was removed and pellets were air dried for 5mins @ RT.

In order to exceed the target concentration (>110ng/μL) needed by BGI, the pellets were resuspended in 500μL (150ng/μL) of EB Buffer (Qiagen). This is assuming each sample has at least 75μg of DNA.

Samples were spec’d on the Roberts Lab NanoDrop1000.

 

Results:

 

Concentrations are on point, the 260/280 ratios are still good and the 260/230 ratios are greatly improved. Samples are ready to send off to BGI for sequencing!

Total yields:

Geoduck: 84μg

Oly: 86μg

Will run these samples out on a gel to verify that the gDNA is still intact and didn’t get degraded during the precipitation.

Agarose Gel – Olympia oyster Whole Body gDNA Integrity Check

Ran the gDNA isolated yesterday from Ostrea lurida whole body on a 0.8% modified TAE gel (w/EtBr) to assess gDNA integrity. Used 1μL of each sample.

 

Results:

The results are not good. Every sample exhibits serious degradation (the smearing that’s present in each lane). There should be a distinct, high molecular weight band with no smearing if the gDNA was high quality and intact. These extractions also served as a comparison in slight differences in the extraction procedure (homogenization with & without mortar/pestle), as described in Steven’s post. However, those differences seem to have no impact on the quality of the resulting gDNA.

I isolated gDNA from Ostrea lurida tissue samples two weeks ago using the E.Z.N.A. Mollusc DNA Kit (Omega Bio-Tek) and didn’t see this level of degradation. Additionally, Katherine Silliman used the E.Z.N.A. Mollusc DNA Kit to isolate gDNA from Ostrea lurida larvae and obtained high quality gDNA in virtually all of her samples. Below is my gel and Katherine’s gel for quick comparison to the one above:

Ostrea lurida gDNA isolated from adductor muscle & mantle tissues (lanes 4 & 5). Despite low quantity loading, notice that smearing below high molecular weight bands is limited to a low molecular weight range.

Katherine’s gel of Ostrea lurida gDNA isolated from larvae.

 

 

 

 

 

 

 

 

 

 

 

 

I can’t be certain what is causing this issue. We previously had this same issue with a different group of Ostrea lurida whole body gDNA isolations (using a DNeasy Blood & Tissue Kit [Qiagen]). Two different kits using whole bodies and both sets of extractions have produced similarly bad results. It’s certainly possible that some nastiness (that’s a scientific term, btw) is being introduced by using whole body instead of specific tissues.

Another possible contributor to the DNA degradation we’ve seen is how the samples were collected and stored. I’m not up-to-date on exactly how the preservation was accomplished, but I do know that the Ostrea lurida whole body samples I previously worked with were just masses of black when I removed them from shells/tubes for isolation. So, in that case, it wasn’t terribly surprising that that the gDNA obtained from those was degraded. It should also be noted that Katherine’s extraction were from whole larvae that had been stored in RNAlater. Although a direct comparison cannot be made due to the difference in developmental stage between Katherine’s samples and these, it lends some evidence to the possibility that sample collection/storage is a contributor to the degraded gDNA we’re obtaining from whole body oyster extractions. However, with that being said, I’m not sure what the collection and storage background is on this particular set of samples.

DNA Isolation – Olympia oyster whole body

Continued the extractions that Steven began yesterday and this morning using the E.Z.N.A. Mollusc DNA Kit (Omega Bio-Tek) after the RNase treatment @ 70C.

  • Samples were cooled to RT (~10mins)
  • Added 300μL of 100% EtOH to each (equivalent to the volume of aqueous phase Steven recovered from each sample)
  • Followed manufacturer’s protocol
  • Eluted all samples with 50μL of Elution Buffer
  • Spec’d on Roberts Lab NanoDrop1000

 

Results:

 

 

 

DNA looks absolutely pristine and has amazing yields.

Will check gDNA integrity via agarose gel tomorrow.

Data has been entered into the master spreadsheet for this project: Ostrea lurida Project Master Tissue Sample List

Agarose Gel – Geoduck gDNA Integrity Check

Ran 0.8% agarose 1x modified TAE gel stained with EtBr to assess the integrity of geoduck gDNA isolated earlier today.

Ran ~500ng of each sample:

Geoduck adductor muscle 1: 4μL

 

Results:

 

 

 

The gDNA looks really good with a prominent high molecular weight band and little smearing.

Will proceed with pooling all accumulated geoduck gDNA for this project.

Genomic DNA Isolation – Geoduck Adductor Muscle & Foot

Just need a tad bit more gDNA for the geoduck genome sequencing project with BGI. Currently have ~69 and need a minimum of 73μg.

Isolated gDNA from Panopea generosa (geoduck) adductor muscle & foot samples collected by Brent & Steven on 20150811 using the DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

  • 124.4mg of adductor muscle 1
  • Tissue homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tube to 1000μL with DNAzol and incubated @ RT for 10mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellet was resuspended in 400μL of Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tube

Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing next week.

 

Results:

 

 

There was a great deal of insoluble material from the get-go that was carried through the entire isolation.

Overall, the 260/280 ratio looks pretty good, but the 260/230 ratio is just trash. As can be seen in the plots above, there is clearly significant absorbance in the 230 – 250nm, suggesting some contaminant carryover (phenol/salt).

Will assess gDNA integrity on a gel.

 

Yield of geoduck gDNA from this isolation: ~48μg

Total accumulated geoduck gDNA for this project: ~117μg

Great! Have sufficient gDNA to send to BGI (minimum of 73μg needed). Assuming gDNA integrity looks good on a gel, I will pool samples tomorrow, quantify the pooled gDNA and prepare for submission.

Agarose Gel – Geoduck & Olympia Oyster gDNA Integrity Check

Ran 0.8% agarose 1x modified TAE gel stained with EtBr to assess the integrity of geoduck gDNA and Olympia oyster gDNA isolated earlier today.

Ran ~500ng of each sample:

Geoduck adductor muscle 1: 6.8μL

Geoduck adductor muscle 2: 20μL (260ng)

Geoduck foot 1: 16.6μL

Geoduck foot 2: 20μL (200ng)

Oly adductor muscle: 4μL

Oly mantle: 4.7μL

Results:

 

 

 

 

 

 

The gel is loaded in the order listed above (going left to right on the gel).

All samples look really good with prominent high molecular weight bands and little smearing.

Genomic DNA Isolation – Olympia oyster adductor musle & mantle

Isolated gDNA from Ostrea lurida (Olympia oyster) adductor muscle & mantle samples collected by Brent & Steven on 20150812 using DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

  • 73.5mg of adductor muscle
  • 146mg of mantle
  • Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tubes to 1000μL with DNAzol and incubated @ RT for 10mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended in 200μL of Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes

Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing.

 

Results:

 

 

There was a great deal of insoluble material from the get-go that was carried through the entire isolation.

Overall, the 260/280 ratios look pretty good, but the 260/230 ratios are just trash. As can be seen in the plots above, there is clearly significant absorbance in the 230 – 250nm, suggesting some contaminant carryover (phenol/salt).

Will evaluate gDNA integrity on agarose gel.

 

Yields from this isolation:

Adductor muscle: 18.75μg

Mantle: 15.9μg

 

Total Olympia oyster gDNA from this isolation: 34.65μg

 

Total Olympia oyster gDNA accumulated for this project: 88.75μg

 

Great! Have sufficient gDNA to send to BGI (minimum of 73μg needed). Assuming gDNA integrity looks good on a gel, I will pool samples tomorrow, quantify the pooled gDNA and prepare for submission.

Genomic DNA Isolation – Geoduck Adductor Muscle & Foot

Isolated gDNA from Panopea generosa (geoduck) adductor muscle & foot samples collected by Brent & Steven on 20150811 using the DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

  • 102.5mg of adductor muscle 1
  • 76.7mg of adductor muscle 2
  • 84.2mg of foot 1
  • 54.5mg of foot 2
  • Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tubes to 1000μL with DNAzol and incubated @ RT for 10mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended in 200μL of Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes

Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing next week.

 

Results:

 

 

There was a great deal of insoluble material from the get-go that was carried through the entire isolation.

Overall, the 260/280 ratios look pretty good, but the 260/230 ratios are just trash. As can be seen in the plots above, there is clearly significant absorbance in the 230 – 250nm, suggesting some contaminant carryover (phenol/salt).

Will evaluate gDNA integrity on agarose gel.

Yields from this isolation:

Adductor muscle 1: 11.03μg

Adductor muscle 2: 1.95μg

Foot 1: 4.6μg

Foot 2: 1.64μg

 

Total geoduck gDNA from this isolation: 19.2μg

 

Total geoduck gDNA accumulated for this project: 69μg

Still need an additional 4μg at a minimum! Will isolate more gDNA tomorrow…

Agarose Gel – Geoduck & Olympia Oyster gDNA Integrity Check

Ran 0.8% agarose 1x modified TAE gel stained with EtBr to assess the integrity of geoduck gDNA and Olympia oyster gDNA isolated yesterday.

Ran ~500ng of each sample:

Geoduck adductor muscle 1: 4.4μL

Geoduck adductor muscle 2: 20μL (355ng)

Geoduck foot 1: 20μL

Geoduckk foot 2: 6.9μL

Oly adductor muscle: 5.5μL

Oly mantle: 3.05μL

Results:

 

 

 

 

The gel is loaded in the order listed above (going left to right on the gel).

All samples look really good with prominent high molecular weight bands and little smearing.

Current total approximate yields from all extractions from both species are as follows:

Geoduck: 49.8μg

Olympia oyster: 54.1μg

Still need ~25μg of each species to have sufficient quantities for sequencing.

Genomic DNA Isolation – Olympia oyster adductor musle & mantle

Previously isolated gDNA from these tissues on 20150901. However, found out after the isolations that BGI needs >73μg of gDNA for the genome sequencing project, which is significantly more than I obtained previously.

Isolated gDNA from Ostrea lurida (Olympia oyster) adductor muscle & mantle samples collected by Brent & Steven on 20150812 using DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

  • 89.8mg of adductor muscle
  • 92.2mg of mantle
  • Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tubes to 1000μL with DNAzol and incubated @ RT for 10mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended in 200μL of Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes

Spec’d on Roberts Lab NanoDrop1000 (ThermoFisher) and stored temporarily at 4C to avoid freeze-thawing before sending off for sequencing.

 

Results:

 

There was a great deal of insoluble material from the get-go that was carried through the entire isolation.

Overall, the 260/280 ratios look pretty good, but the 260/230 ratios are just trash. As can be seen in the plots above, there is clearly significant absorbance in the 230 – 250nm, suggesting some contaminant carryover (phenol/salt).

Will evaluate gDNA integrity on agarose gel.

Total yield from this isolation is still below the minimum quantity of gDNA needed for the sequencing project. Will need to perform another round of gDNA isolation.