Pooled all of the geoduck gDNA from all the previous geoduck isolations for the Geoduck Genome Sequencing Project and pooled all of the Ostrea lurida gDNA from previous Ostrea lurida isolations for the Olympia Oyster Genome Sequencing Project.
Both sets of gDNA were ethanol (EtOH) precipitated. This was done for two reasons - to concentrate the samples to the minimum necessary concentration required by BGI (>119ng/μL) and to try to improve the poor 260/230 ratios (which are likely due to high salt carryover).
Precipitation was performed by consolidating each species of DNA in 15mL conicals, adding 0.1 volumes of 3M sodium acetate (pH= 5.2) and then adding 2.5 volumes of ice cold (-20C) 100% EtOH. Volumes used are below.
| Sample | DNA Vol (μL) | 3M Sodium Acetate Vol. (μL) | 100% EtOH Vol (μL) | Total Vol (μL) |
| Geoduck | 1860 | 186 | 5115 | 7161 |
| Oly | 780 | 78 | 2145 | 3003 |
Samples were mixed by inversion and incubated @ -80C for 3hrs.
DNA was pelleted by spinning tubes at 12,000g for 30mins @ 4C in a SL-50T (Sorval) rotor in a T21 (Sorval) table top centrifuge.
Supernatant was decanted and pellets were transferred to 1.5mL snap cap tubes.
Pellets were washed three times with 70% EtOH.
After the last wash, the supe was removed and pellets were air dried for 5mins @ RT.
In order to exceed the target concentration (>110ng/μL) needed by BGI, the pellets were resuspended in 500μL (150ng/μL) of EB Buffer (Qiagen). This is assuming each sample has at least 75μg of DNA.
Samples were spec’d on the Roberts Lab NanoDrop1000.
Results:
(http://eagle.fish.washington.edu/Arabidopsis/20150917_gDNA_geoduck_oly_ODs.JPG)
(http://eagle.fish.washington.edu/Arabidopsis/20150917_gDNA_geoduck_oly_plots.JPG)
Concentrations are on point, the 260/280 ratios are still good and the 260/230 ratios are greatly improved. Samples are ready to send off to BGI for sequencing!
Total yields:
Geoduck: 84μg
Oly: 86μg
Will run these samples out on a gel to verify that the gDNA is still intact and didn’t get degraded during the precipitation.