Phenol-Chloroform DNA Clean Up - Mac and Claire’s Samples (from 20140410)

2014
Lineage-specific DNA methylation patterns in developing oysters
Author

Sam White

Published

April 14, 2014

Due to low 260/230 values and Mac’s smeary sample, performed a phenol-chloroform DNA cleanup on the samples isolated 20140410.

  1. Brought volume of each sample to 200uL with Buffer EB (Qiagen).

  2. Added an equal volume (200uL) of 25:24:1 Phenol/Chloroform:Isoamyl alcohol.

  3. Mixed on rotator for 20mins @ RT.

  4. Separated aqueous/organic phases by spinning at 12,000g 5mins @ RT.

  5. Transferred aqueous phase to new tube. Repeated steps 2-4 until samples exhibited no more interphase. Combined aqueous phases in to a single tube for each of the two samples.

  6. Added and equal volume of chloroform (170uL).

  7. Mixed on rotator for 20mins @ RT.

  8. Separated aqueous/organic phases by spinning at 12,000g 5mins @ RT.

  9. Transferred aqueous phase to new tube.

Performed an ethanol precipitation on each sample.

  1. Added 0.1 volumes of 5M sodium acetate (pH = 5.2).

  2. Added 2 volumes of ice cold 100% EtOH.

  3. Incubated 20mins @ -20C.

  4. Pelleted DNA by spinning 16,000g, 20mins @ 4C.

  5. Discarded supe and washed pellets with 1mL 70% EtOH.

  6. Pelleted DNA by spinning 16,000g, 5mins @ 4C.

  7. Repeated steps 5-6 one time.

  8. Removed all supernatant and resuspended in 100uL of nuclease-free H2O.

  9. Spec’d on NanoDrop1000.

NOTE: Mac’s sample exhibited the same chunky/cloudiness upon addition of 100% EtOH that has been seen previously by both her and myself…

Results:

So, the clean up seemed to work wonders on the 260/230 values. Not surprisingly, Mac’s sample didn’t clean up nearly as nicely as Claire’s, based on my observations of the odd behavior during EtOH precipitation.

And, despite the nice, clean looking peaks, the 260/280 ratios are actually WORSE than the original isolation. Will run on gel for a further assessment of quality/integrity.

Loaded 5uL of each sample (~600ng) on a 1.0% agarose, 1x modified TAE gel stained with ethidium bromide.

Gel Layout:

Lane 1 - Hyperladder I (Bioline)

Lane 2 - Claire’s CgF gonad sample

Lane 3 - Mac’s gonad sample

Used Hyperladder I this time, which has a high molecular weight band of 10kb and a low molecular weight band of 200bp.

Well, this totally sucks. Both samples appear to consist of nothing but 150-200bp fragments. Is something actually degrading these samples? The Buffer EB I used during the initial extraction is certainly old. Possible source of degradation? Ugh. Maybe I’ll try this again, but resuspend in TE…