Tag Archives: phenol-chloroform cleanup

Phenol:Chloroform Extractions and EtOH Precipitations – MspI Digestions of C.virginica DNA from Earlier Today

The two MspI restriction digestions from earlier today for our project with Qiagen were subjected to phenol:chloroform cleanup and subsequent ethanol precipitations.

Phenol:chloroform clean up procedure:

  1. Added equal volume (50μL) of phenol:chloroform:IAA (25:24:1) to each sample.

  2. Vortexed.

  3. Centrifuged 5mins, 16,000g at room temperature.

  4. Transferred aqueous phase (top layer) to clean 0.5mL snap-cap PCR tube.

  5. Added equal volume of chloroform (50μL) to aqueous phase.

  6. Vortexed.

  7. Centrifuged 5mins, 16,000g at room temperature.

  8. Transferred aqueous phase (top layer) to clean 0.5mL snap-cap PCR tube.

Performed ethanol precipitation on both samples according to lab protocol.

Resuspended precipitated DNA in 25μL Buffer EB (Qiagen).

Will quantify with Qubit 3.0.

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Phenol-Chloroform DNA Cleanup – Geoduck gDNA

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.

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Phenol-Chloroform DNA Cleanup – Olympia Oyster gDNA

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 (380μL) of chlforoform:isoamyl alcohol (24:1)
  • Mixed by hand – moderate shaking
  • Centrifuged 2mins, 10,000g, RT
  • Transferred aqueous phase (320μL) to clean tube
  • Added 0.1vols (32μL) of 3M sodium acetate (pH = 5.2)
  • Added 2vols (640μL) 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 547.15 200 109,430
Quant-IT 74.26 200 14,851

 

The NanoDrop1000 overestimates the concentration of the sample by 7.4x! That’s really insane!

Regardless, this is a solid yield (using yield from Quant-IT) and, when combined with the other Ostrea lurida gDNA that I isolated 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

The clean up seems to have worked well, as the absorbance spectrum is much improved and nearly mirrors that of the Oly gDNA isolated with the Mollusc Kit.

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

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…

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Restriction Digestions – HpaII and MspI on Mac’s C.gigas Samples: Round 2

Continued with 2nd round of digestions from yesterday. All samples were resuspended in 25uL of H2O yesterday, so brought volume up to 44uL with H2O, added 5uL of appropriate 10X Buffer (HpaII = NEB Buffer #4, MspI = NEB Buffer #1), added 1uL of enzyme, incubated 37C for 3hrs. Heat-inactivated all samples @ -80C for 30 mins.

Phenol:Chloroform Extractions

Restriction digests  were mixed with equal volume (50uL) of phenol:chloroform:IAA (25:24:1) and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to a clean tube and an equal volume (50uL) of chloroform was added. Samples were mixed and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to clean tubes and stored @ -20C. Will EtOH precipitate and spec on Monday.

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Phenol:Chloroform Extractions and EtOH Precipitations – HapII and MspI digests from yesterday

Restriction digests from yesterday were mixed with equal volume (50uL) of phenol:chloroform:IAA (25:24:1) and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to a clean tube and an equal volume (50uL) of chloroform was added. Samples were mixed and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to clean tubes and EtOH precipitated, according to protocol. Samples were resuspended in 25uL of H2O and spec’d.

Samples are labeled as Parent (P), #, tissue, enzyme (MspI = M, HpaII = H, Undigested = U)

Results:

Here is a link to a spreadsheet with ODs. Average recovery was ~734ng, which is only a 36% recovery (started with 2000ng). Will need to discuss with Mac and Steven to see if it’s worth continuing with these sample through a second round of digests/phenol:chloroform extraction/EtOH precipitation, as I don’t know what quantity of DNA is required/desired for the subsequent methylation specific PCR (MSP), OR if I should/need to perform a repeat of these 1st-round digestions in order to end up with sufficient DNA for MSP.

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