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:
- 102.5mg of adductor muscle 1
- 76.7mg of adductor muscle 2
- 84.2mg of foot 1
- 54.5mg 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.
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 1: 11.03μg
Adductor muscle 2: 1.95μg
Foot 1: 4.6μg
Foot 2: 1.64μg
Total geoduck gDNA from this isolation: 19.2μg
Total geoduck gDNA accumulated for this project: 69μg
Still need an additional 4μg at a minimum! Will isolate more gDNA tomorrow…