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:
- 73.5mg of adductor muscle
- 146mg 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:
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.
Yields from this isolation:
Adductor muscle: 18.75μg
Mantle: 15.9μg
Total Olympia oyster gDNA from this isolation: 34.65μg
Total Olympia oyster gDNA accumulated for this project: 88.75μg
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.