Tag Archives: EtOH precipitation

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.

MBD Enrichment – Crassostrea virginica Sheared DNA Day 3

Continued MBD enrichment of C.virginica DNA from yesterday for Qiagen project.

Followed the MethylMiner Methylated DNA Enrichment Kit (Invitrogen) manufacturer’s protocol for input DNA amounts of 1 -10ug (I am using 8ug in each of two samples).

Since the protocol has two elution steps that are each saved separately from each other for each sample, I did the following to combine the two elution fractions into a single sample:

  • Pelleted one elution fraction from each sample
  • Discarded supernatant from pelleted sample
  • Transferred second elution fraction to the pellet from the first elution fraction
  • Pelleted second elution fraction

The rest of the ethanol precipitation procedure was followed per the manufacturer’s protocol.

Final pellets were resuspended in 25μL of Buffer EB (Qiagen) and stored temporarily on ice for quantification.

Ethanol Precipitation – Olympia oyster MBD

Precipitated the MBD enriched DNA from yesterday according to the MethylMiner Methylated DNA Enrichment Kit (Invitrogen) protocol.

However, since the protocol has two elution steps that are each saved separately from each other for each sample, I did the following to combine the two elution fractions into a single sample:

  • Pelleted one elution fraction from each sample
  • Discarded supernatant from pelleted sample
  • Transferred second elution fraction to the pellet from the first elution fraction
  • Pelleted second elution fraction

The rest of the ethanol precipitation procedure was followed per the manufacturer’s protocol.

Final pellets were resuspended in 25μL of Buffer EB (Qiagen) and stored @ 4C.

MBD enriched DNA will be quantified tomorrow.

Ethanol Precipitation – Geoduck & Olympia oyster gDNA

Pooled all of the geoduck gDNA from all the previous geoduck isolations for the Geoduck Genome Sequencing Project and pooled all of the Ostrea lurida gDNA from previous Ostrea lurida isolations for the Olympia Oyster Genome Sequencing Project.

Both sets of gDNA were ethanol (EtOH) precipitated. This was done for two reasons – to concentrate the samples to the minimum necessary concentration required by BGI (>119ng/μL) and to try to improve the poor 260/230 ratios (which are likely due to high salt carryover).

Precipitation was performed by consolidating each species of DNA in 15mL conicals, adding 0.1 volumes of 3M sodium acetate (pH= 5.2) and then adding 2.5 volumes of ice cold (-20C) 100% EtOH. Volumes used are below.

Sample DNA Vol (μL) 3M Sodium Acetate Vol. (μL) 100% EtOH Vol (μL) Total Vol (μL)
Geoduck 1860 186 5115 7161
Oly 780 78 2145 3003

Samples were mixed by inversion and incubated @ -80C for 3hrs.

DNA was pelleted by spinning tubes at 12,000g for 30mins @ 4C in a SL-50T (Sorval) rotor in a T21 (Sorval) table top centrifuge.

Supernatant was decanted and pellets were transferred to 1.5mL snap cap tubes.

Pellets were washed three times with 70% EtOH.

After the last wash, the supe was removed and pellets were air dried for 5mins @ RT.

In order to exceed the target concentration (>110ng/μL) needed by BGI, the pellets were resuspended in 500μL (150ng/μL) of EB Buffer (Qiagen). This is assuming each sample has at least 75μg of DNA.

Samples were spec’d on the Roberts Lab NanoDrop1000.

 

Results:

 

Concentrations are on point, the 260/280 ratios are still good and the 260/230 ratios are greatly improved. Samples are ready to send off to BGI for sequencing!

Total yields:

Geoduck: 84μg

Oly: 86μg

Will run these samples out on a gel to verify that the gDNA is still intact and didn’t get degraded during the precipitation.

EtOH Precipitation – LSU C.virginica Oil Spill MBD Continued (from 20141126)

Precipitation was continued according to the MethylMiner Methylated DNA Enrichment Kit (Invitrogen). Since I will need sample volumes of 24uL for the subsequent bisulfite conversion, I resuspended the samples in 29uL of water (will use 2.5uL x 2 reps for quantification).

Samples to be quantified:

NC = non-captured (i.e. non-methylated)

E = eluted (i.e. methylated)

  • HB2 NC
  • HB5 NC
  • HB16 NC
  • HB30 NC
  • NB3 NC
  • NB6 NC
  • NB11 NC
  • NB21 NC
  • HB2 E
  • HB5 E
  • HB16 E
  • HB30 E
  • NB3 E
  • NB6 E
  • NB11 E
  • NB21 E
  • Control NC
  • Control E

Samples were quantified using the Quant-IT BS Kit (Invitrogen) with a plate reader (BioTek). All samples were run in duplicate. Used 2.5uL of each sample for quantification.

Samples were stored in @ -20C (FTR 209) in the bisulfite seq box created by Claire for this project.

Results:

20141202_LSU_Virginica_MBD:

https://docs.google.com/spreadsheets/d/1NrrVmYsUQcstnrt4583mYN2PeVav54luyFvVUEkcjWE/edit?usp=sharing

Ethanol Precipitation – Colleen’s Sea Star Coelomycete RNA from Yesterday

Performed an EtOH precipitation on the sea start RNA due to some residual column resin (?) in the tubes after elution.

Added 0.1 volumes of 3M sodium acetate (pH=5.2; 10uL), 1uL glycogen (Ambion stock 5mg/mL), 2.5 volumes of ice cold 100% EtOH (275uL). Vortexed and incubated O/N at -20C.

Pelleted RNA 16,000g, 30mins @ 4C.

Discarded supe.

Washed pellet 70% EtOH.

Pelleted RNA 16,000g, 15mins @ 4C.

Repeated wash and spin.

Removed supe, resuspended RNA in 50uL of 0.1%DEPC-treated H2O and spec’d on NanoDrop1000.

Samples were stored in Shellfish RNA Box #5.

Results:

Well, overall, the RNA looks immensely better than yesterday. However, as expected, there has been some slight loss with all the additional manipulations. As such, yields are low (although, they were initially low, too). However, I think most of the samples will be usable, albeit bordering on the minimum amount of total RNA needed (200ng) at the Cornell sequencing facility…

DNA Precipitation – Geoduck DNA from 20140213

After speaking with Axa regarding the DNA concentrations, he would like the DNA from the ethanol-fixed tissue to be more concentrated, and he wants them in ddH2O instead of Buffer AE (from the Qiagen DNeasy Kit). So, I preformed a standard ethanol precipitation. Added 0.1 volumes of 3M sodium acetate (pH = 5.2) [15uL], 2.5 volumes of 100% ethanol [412.5uL] and incubated @ -20C over the weekend.

Pelleted DNA by spinning 16,000g, 15mins @ 4C. Discarded supe, washed pellets with 1mL 70% ethanol and re-pelleted the DNA. Performed a second wash with 70% EtOH, pelleted DNA, discarded supe, resuspended DNA in 75uL of NanoPure H2O, and spec’d on NanoDrop1000.

Results:

Will spec when I re-isolate DNA from “fresh” geoduck samples for coordinating Axa’s DNA sequencing project with our potential RNA-seq project. See 20140219.

RNA Isolation – C.gigas Larvae from 20110412 & 20110705 (Continued from 20120112)

All of the RNA samples were re-combined with their respective counterparts and subject to a standard EtOH precipitation (0.1 volumes of 3M NaOAc, pH = 5.2, 2.5 volumes 100% EtOH; incubated -80C 1hr; pelleted; washed with 1mL 70% EtOH; pelleted). Pellets were washed two additional times (for a total of three washes) with 70% EtOH. RNA was resuspended in 50uL of 0.1% DEPC-H2O and spec’d on the Roberts Lab NanoDrop 1000.

Results:

Yields for the 4/12/2011 samples were all lower than the yields for the 7/5/2011 samples. However, the RNA quality (based on OD260/280 ratios) looks pretty good for both groups of RNA. RNA will be treated with DNase before reverse transcription.

Ethanol Precipitation – Full-length PGS1 cDNA (from 20110921)

Performed an EtOH on gel-purified PCR products from 20110921. Briefly, added 0.1 vols of 3M sodium acetate (pH=5.2; 43uL), mixed and then added 2.5 vols of 100% EtOH (1182.5uL). Mixed, split into two tubes (due to high volume not fitting in a single tube) and incubated @ -80C O/N. Pelleted DNA 16,000g, 20mins, 4C. Discarded supe. Washed pellet w/ 1mL 70% EtOH. Pelleted DNA 16,000g, 15mins, 4C. Discarded supe. Resuspended both pellets in a TOTAL of 25uL Qiagen Buffer EB (10mM Tris-HCl) and spec’d.

Results:

Now have sufficient DNA for sequencing.

What’s next?

Generate PCR product using primers that anneal OUTSIDE of each of the qPCR primers and then sequence those bands to ensure that the qPCR primers are actually annealing to two different isoforms.