Since we still don’t have sufficient gDNA for the full scope of the Olympia oyster genome sequencing, I isolated more gDNA.
Isolated gDNA from 118mg outer mantle tissue collected by Steven & Brent on 20150812.
Tissue was thoroughly minced with a clean razor blade and then processed with the E.Z.N.A. Mollusc Kit (Omega BioTek) with the following changes:
- Doubled solution volumes for steps before sample was loaded on columns
- Sample was split equally in two tubes prior to addition of 100% EtOH
- All mixing was done by shaking – no vortexing! Done this way to, hopefully, maintain gDNA integrity
- Elution volume = 50μL
- Elution was repeated using the initial elution to maximize recovery while maintaining low sample volume.
- The two preps were pooled – final volume = 79μL
DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit
For the Quant-IT kit, the samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.
Standards were run in triplicate, samples were run in duplicate.
96-well black (opaque) plate was used.
Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).
|METHOD||CONCENTRATION (ng/μL)||VOLUME (μL)||YIELD (ng)|
The NanoDrop1000 overestimates the concentration of the sample by 2.5x!
Regardless, this is a solid yield and, when combined with the other Ostrea lurida gDNA that I cleaned up today, should push the total amount of gDNA submitted to BGI over the required threshold.
Will evaluate gDNA quality on a gel.
Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants
NanoDrop1000 Measurements and Plots