Tag Archives: Fidalgo 2SN

DNA Quality Assessment – Geoduck, Oly & Oly 2SN

I recently ran gDNA isolated for geoduck and Olympia oyster genome sequencing, as well as gDNA isolated from the Olympia oyster reciprocal transplant experiment out on a Bioanalyzer (Agilent) using the DNA 12000 chips. The results from the chip were a bit confusing and difficult to assess exactly what was going on with the DNA.

So, I ran 5μL of each of those samples on a 0.8% agarose 1x modified TAE gel w/EtBr to get a better look at how the samples actually looked.

Results:

 

Both the geoduck and the Olympia oyster samples for genome sequencing show intact, high molecular weight bands with some smearing (i.e. degradation). The oly sample looks a bit funky, most likely due to a gel anomaly. I’ll quantify these using a dye-based method for a more accurate quantification before sending off to BGI.

The Fidalgo 2SN samples all have intact, high molecular weight bands, but most of the samples show extensive smearing (i.e. degradation). However, sample 2SN 35 has no visible DNA at all.

Here’s a table highlighting the differences between the Fidalgo gDNA samples:

Sample Fresh/Frozen Isolator
10 Fresh Sam
11 Fresh Sam
12 Fresh Sam
20 Fresh Mrunmayee
21 Fresh Mrunmayee
22 Fresh Mrunmayee
32 Frozen Sam
33 Frozen Sam
34 Frozen Sam
35 Frozen Sam

 

The fresh ctenidia samples were isolated by me on 20151021 and by Mrunmayee on 20151023. The frozen ctenidia samples were isolated by me on 20151103.

It’s interesting to note that Mrunmayee’s isolations appear to exhibit the least amount of degradation. Besides her handling the samples, the primary difference is that her samples were incubated in the buffer/Pro K solution O/N @ 37C, while my fresh samples were incubated @ 60C for 3hrs and my frozen samples were incubated @ 60C for 1hr. Overall, though, the frozen samples look the worst.

Finally, it’s also interesting to see that the two samples isolated using DNazol (geoduck and Olympia oyster genome samples) migrate more slowly than the remaining Olympia oyster samples which were isolated with the E.Z.N.A. Mollusc Kit.

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DNA Quantification & Quality Assessment – Oly 2SN gDNA

Comparison of three different approaches to using the E.Z.N.A. Mollusc Kit:

  • Fresh isolations by me
  • Fresh isolations by Mrunmayee
  • Isolations from tissue frozen in buffer by me

Results:

Bioanalyzer Data File (XAD file): 2100 expert_DNA 12000_DE72902486_2015-11-04_15-06-32.xad

There’s a LOT going on here. Will update this entry tomorrow with more info.

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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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Oyster Sampling – Oly Fidalgo 2SN, 2HL, 2NF Reciprocal Transplants Final Samplings

The remaining Olympia oysters from Jake Heare’s reciprocal transplant experiment have been retrieved from field sites and are awaiting sampling. The oysters have been stored in the cold room (temp?) for 15 days so far.

The previous sampling scheme was described here: DNA Isolations – Fidalgo 2SN Reciprocal Transplants Final Samplings

Sampling scheme for today was as follows:

  1. Assign unique number to oysters (1-100 for each of the three populations)
  2. Photograph with ruler for future shell measurements
  3. Weigh oysters
  4. Dissect ctenidia for DNA isolation in 350μL MBL1 Buffer + 25μL Proteinase K (reagents part of the E.Z.N.A. Mollusc Kit [Omega BioTek)
  5. Preserve portion of remaining body tissue (not viscera; gonad/digestive gland) in 1mL RNAlater (Life Technologies)

Ctenidia samples were stored -80C in the buffer/pro k solution for DNA isolation at a later date.

RNAlater samples will be stored over the weekend at 4C and then transferred to -20C for long term storage.

All oyster data is here (Google Sheet): Oly reciprocal final sampling

All photos from today’s sampling are here: Oyster Measurement Photos

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DNA Isolations – Fidalgo 2SN Reciprocal Transplants Final Samplings

The remaining Olympia oysters from Jake Heare’s reciprocal transplant experiment have been retrieved from field sites and are awaiting sampling. The oysters have been stored in the cold room (temp?) for 6 days so far.

Sampling scheme is as follows:

  1. Assign unique number to oysters
  2. Photograph with ruler for future shell measurements
  3. Weigh oysters
  4. Dissect ctenidia for DNA isolation
  5. Dissect & discard viscera (e.g. digestive gland and gonad)
  6. Weigh remaining body
  7. Preserve remaining body in RNAlater
  8. Weigh empty shells


Mrunmayee photographed & initiated dissections of oysters #3 – 8
. I took over for oyster #9 -14.

All oyster data is here (Google Sheet): Oly reciprocal final sampling

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

  • No optional steps were performed
  • Ctenidia tissue was lysed for 3hrs @ 60C
  • Single elution of 50μL

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

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