Sam's Notebook

Phenol-Chloroform DNA Cleanup - Geoduck gDNA

by ["kubu4"] 1 min read

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)