Just need a tad bit more gDNA for the geoduck genome sequencing project with BGI. Currently have ~69 and need a minimum of 73μg.
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:
- 124.4mg of adductor muscle 1
- Tissue homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
- After homogenization, topped off tube 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
- Pellet was resuspended in 400μL of Buffer EB (Qiagen)
- Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tube
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 ratio looks pretty good, but the 260/230 ratio is 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 assess gDNA integrity on a gel.
Yield of geoduck gDNA from this isolation: ~48μg
Total accumulated geoduck gDNA for this project: ~117μg
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.