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:

(http://eagle.fish.washington.edu/Arabidopsis/20150914_gDNA_Oly_ODs.JPG)

(http://eagle.fish.washington.edu/Arabidopsis/20150914_gDNA_Oly_plots.JPG)

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.