Tag Archives: O’geneRuler DNA Ladder Mix

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.

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.

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.

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.

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 (from 20150828) and Olympia oyster gDNA (from 20150901).

Ran 500ng of each sample:

Geoduck adductor muscle: 4.87μL

Geoduck foot: 4.50μL

Oly adductor muscle: 4.22μL

Oly mantle: 2.51μL

Results:

 

 

The gel was loaded in the same order as the sample volumes listed above.

The gel is a bit disappointing.

Geoduck adductor

High molecular weight band present, but extremely faint. Not too much smearing, but accumulation of low molecular weight smudge suggests residual RNA. This is despite the fact that the kit used for isolation (E.Z.N.A. Mollusc DNA Kit) has a RNase treatment. This residual RNA could explain why the amount loaded on the gel appears to be so little compared to other samples (the RNA is contributing to the absorbance at 260nm, thus inflating the calculated concentration of gDNA).

Geoduck foot

High molecular weight band present and bright. Some smearing (i.e. degradation) present, along with degarded DNA visible at ~500bp. This sample may require a Bioanalyzer run to accurately quantify the high molecular weight DNA, as the lower molecular weight DNA (i.e. degarded DNA) is “artificially” inflating the concentration of the DNA in the sample.

Oly adductor

High molecular weight band present and bright. Some smearing (i.e. degradation) present, along with degarded DNA visible at ~500bp. This sample may require a Bioanalyzer run to accurately quantify the high molecular weight DNA, as the lower molecular weight DNA (i.e. degarded DNA) is “artificially” inflating the concentration of the DNA in the sample.

Oly mantle

High molecular weight band present, but extremely faint. Some smearing and a smudge around 500bp, indicating degraded DNA. The low visibility of this sample on the gel suggests that the concentration determined by the NanoDrop1000 is inaccurate. However, unlike the geoduck adductor sample, it doesn’t appear that RNA carryover is responsible, as there is no noticeable low molecular weight (~100bp) smudge.

 

Overall, the geoduck foot and the Oly adductor samples are likely usable. Currently awaiting clarification from BGI for DNA quantity requirements for the genome sequencing of these two species.