Tag Archives: gDNA

gDNA Isolation – Geoduck Adductor Muscle

My isolation on Friday didn’t yield a sufficient quantity of gDNA for the additional DNA needed for the geoduck genome sequencing project. Used two adductor muscles (Box 1) samples collected by Brent & Steven on 20150811.

Tissue weights:

  • Geoduck adductor 1: 433mg (gone)
  • Geoduck adductor 2: 457.4mg (gone)

 

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

 

  • Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tubes to 960μL with DNAzol, 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)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes

NOTE: Both samples produced a stark white, “cottony” precipitate after the addition of the ethanol. This precipitate was transferred to a clean tube and processed in the same fashion.

 

Resuspension volumes

Adductor 1:  200μL

Adductor 2: 50μL

Adductor 1 & 2 fluff: 500μL each

 

Spec’d on Roberts Lab NanoDrop1000.

Results:

 

NOTE: The sample labeled “gDNA geoduck adductor 1″ is actually adductor 2. The sample labeled “gDNA geoduck adductor 1{1} is actually adductor 1. However, this is probably moot since these two samples will be pooled shortly.

I’m not going to speculate why there’re weird peaks at 240nm…

The two “fluff” samples aren’t good (extremely high 260/280 ratios, very low 260/230 ratios, and weird peak at 240nm). Not sure what the fluff is that precipitated out with the EtOH addition. Will discard them.

The two normal samples look fine. Will use them for pooling.

Yields

Adductor 1: 52.2μg

Adductor 2: 8.25μg

Restriction Digest – Oly gDNA for RAD-seq w/AlfI

Used a subset (10 samples) from the Ostrea lurida gDNA isolated 20150916 to prepare RAD libraries. This will be done to assess whether or not these samples, which appear to be heavily degraded, are viable for RAD-seq.

Followed the 2bRAD protocol (PDF) developed by Eli Meyer’s lab.

Prepared 1.2μg of each of the following samples in a volume of 10μL:

Google Sheet: 20150930_RADseq_DNA_calcs

 

Prepared a 150μM working stock of the SAM buffer needed for the restriction digestion by diluting 30μL of the supplied stock (500μM) in 70μL NanoPure H2O (total volume = 100μL). This working stock was stored @ -20C in FTR 209 in the “RAD-seq Reagents” box.

Prepared master mix for restriction enzyme reaction:

REAGENT SINGLE REACTION (μL) x11
DNA 8 NA
10x Buffer R 1.2μL 13.2μL
150μM SAM 0.8μL 8.8μL
AlfI 0.5μL 5.5μL
H2O 1.5μL 16.5μL

 

Combined 4μL of the master mix with 8μL of each sample in 0.5mL snap cap tubes. Incubated @ 37C O/N in thermal cycler (no heated lid).

Sample Submission – Olympia Oyster gDNA for Genome Sequencing @ BGI

Shipped the pooled gDNA we’ve been accumulating to BGI to initiate the Olympia oyster genome sequencing project.

Sample was shipped on dry ice with the appropriate paperwork required by BGI (sample declaration letter).

Assigned BGI Lot: 1509191001

Agarose Gel – Geoduck & Olympia oyster gDNA Integrity Check

Ran a 0.8% agarose, 1x modified TAE gel (w/EtBr) with geoduck and Olympia oyster gDNA that was precipitated earlier today. Used 5μL of each sample (~500ng).

Results:

Geoduck gDNA on left. Oly gDNA on right.

 

 

 

 

 

 

 

 

 

 

 

 

 

Overall, the DNA still looks very good. Slight smearing (indicating slight degradation), but the high molecular weight band is very prominent. Will fill out the necessary BGI forms and ship samples out on Monday.

 

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.