The gDNA I extracted on 20151104 didn’t look great on the NanoDrop so I decided to perform a phenol-chloroform cleanup to see if I could improve the NanoDrop1000 absorbance spectrum and, in turn, the quality of the gDNA.
Added an equal volume (500μL) of phenol:chloroform:isoamyl alcohol (25:24:1) to the DNA
Mixed by hand - moderate shaking
Centrifuged 2mins, 10,000g, RT
Transferred aqueous phase to clean tube and discarded interphase & organic phase
Added an equal volume (280μL) of chlforoform:isoamyl alcohol (24:1)
Mixed by hand - moderate shaking
Centrifuged 2mins, 10,000g, RT
Transferred aqueous phase (210μL) to clean tube
Added 0.1vols (21μL) of 3M sodium acetate (pH = 5.2)
Added 2vols (420μL) of 100% EtOH
Mixed by inversion
Incubated @ -20C, 1hr (probably not necessary since gDNA clearly precipitated out as soon as I mixed the sample)
Pelleted DNA by centrifuging 15mins, 12,000g, RT
Discarded supe
Washed pellet with 1000μL cold (-20C) 70% EtOH
Centrifuged 5mins, 12,000g, RT
Discarded supe
Repeated was steps three more times
Resuspended pellet in 100μL of Buffer EB (Qiagen)
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).
Results:
| METHOD | CONCENTRATION (ng/μL) | VOLUME (μL) | YIELD (ng) |
| NanoDrop1000 | 371.83 | 100 | 37,183 |
| Quant-IT | 100.83 | 100 | 10,082 |
The NanoDrop1000 overestimates the concentration of the sample by 3.7x!
Regardless, the yield isn’t all that great (using yield from Quant-IT), which has generally been the case for all of the geoduck gDNA isolations I’ve performed. It would probably be prudent to try isolating gDNA from a different tissue to see if yields improve…
Will evaluate gDNA quality on a gel.
Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants
NanoDrop1000 Measurements and Plots
The clean up procedure didn’t really seem to help with the geoduck sample, as we’re still seeing a significant amount of absorbance from 230 - 250nm.
(http://eagle.fish.washington.edu/Arabidopsis/20151124_gDNA_geoduck_oly_ODs.JPG)
(http://eagle.fish.washington.edu/Arabidopsis/20151124_gDNA_geoduck_oly_plots.JPG)