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.