Tag Archives: foot

DNA Isolation – Geoduck & Olympia Oyster

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

Also need to evaluate DNA quality of initial broodstock samples from Jake’s Olympia oyster reciprocal transplant experiment. Used mantle samples stored in EtOH collected by Hannah (see her notebook entries on July 25 & Sept 5, 2013)

Tissue weights:

  • Geoduck foot: 108.5mg (gone)
  • Olympia oyster adductor: 258.7mg (gone)
  • Oly NF1A: 7.1mg (gone)
  • Oly SN49A: 20.8mg

 

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

 

Genome sequencing resuspension volumes: 50μL

Oly reciprocoal resuspension volumes: 25μL

Spec’d on Roberts Lab NanoDrop1000.

Results:

 

Genome Sequencing Samples

The 260/280 ratios look fine. The 260/230 ratios look poor, as is usually the case after DNAzol isolations.

Yields:

Geoduck: 7.6μg

Oly: 16.5μg

The geoduck yield is insufficient to make up the quantity of gDNA still needed by BGI for sequencing. Will have to isolate more gDNA on Monday.

 

Reciprocal Transplant Samples

The 260/280 ratios look fine. The 260/230 ratios look poor, as is usually the case after DNAzol isolations.

Yields:

NF1A: 7,1μg

SN49A: 1.375μg

The yields are surprisingly good! Next up is to evaluate the gDNA quality on a gel to see if the samples from this experiment will be usable.

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Genomic DNA Isolation – Geoduck Adductor Muscle & Foot

Previously isolated gDNA from these tissues on 20150828. 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 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:

  • 58.8mg of adductor muscle 1
  • 84.0mg of adductor muscle 2
  • 70.3mg of foot 1
  • 95.1mg 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). Oddly, the side-by-side isolations from two different collections of the same tissue type yielded drastically different quantities of gDNA than each other.

Will evaluate gDNA integrity on agarose gel.

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

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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 E.Z.N.A Mollusc DNA Kit (Omega Bio-Tek) according to the manufacturer’s protocol, with the following adjustments:

  • 41.8mg of adductor muscle
  • 30.0mg of foot used
  • Tissues homogenized in 350μL of ML1 Buffer with disposable mortar/pestle tubes using only three pestles strokes
  • Homogenized tissue incubated in ML1 Buffer + Proteinase K @ 60C for 2.5hrs
  • Added 265μL of MBL Buffer
  • Added 514μL of 100% EtOH.
  • Eluted with 75μL Elution Buffer.

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

 

Results:

 

 

 

Yields are good (~7ug).

Quality (260/280 ratios) looks great for both samples.

260/230 ratio not very good for adductor muscle, but perfect for foot tissue.

Will run samples on gel to assess gDNA integrity.

 

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Bioanalyzer Data – Geoduck RNA from Histology Blocks

I received the Bioanalyzer data back for the geoduck foot RNA samples I submitted 20150422. The two samples were run on the RNA Pico chip assay.

 

Results:

Bioanalzyer 2100 Data File (XAD): SamWhite_Eukaryote Total RNA Pico_2015-04-23_13-04-16.xad

Data file requires 2100_Expert_B0208_SI648_SR2 version of the software (Windows).

Gel Representation

 

Electropherogram

 

The samples look really good! As we’ve seen previously in shellfish RNA, there is a single, prominent rRNA band/peak with very little degradation (smearing and/or no prominent peak/band).

Will proceed with RNA isolation from the remaining histology blocks.

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