Tag Archives: DNA Isolation

DNA Isolation & Quantification – Metagenomics Water Filters

After discussing the preliminary DNA isolation attemp with Steven & Emma, we decided to proceed with DNA isolations on the remaining 0.22μm filters.

Isolated DNA from the following five filters:

DNA was isolated with the DNeasy Blood & Tissue Kit (Qiagen), following a modified version of the Gram-Positive Bacteria protocol:

  • filters were unfolded and unceremoniously stuffed into 1.7mL snap cap tubes
  • did not perform enzymatic lysis step
  • filters were incubated with 400μL of Buffer AL and 50μL of Proteinase K (both are double the volumes listed in the kit and are necessary to fully coat the filter in a 1.7mL snap cap tube)
  • 56oC incubations were performed overnight
  • 400μL of 100% ethanol was added to each after the 56oC incubation
  • samples were eluted in 50μL of Buffer AE
  • all spins were performed at 20,000g

Samples were quantified with the Roberts Lab Qubit 3.0 and the Qubit 1x dsDNA HS Assay Kit.

Used 5μL of each sample for measurement (see Results for update).

Results:

Raw data (Google Sheet): 20180426_qubit_metagenomics_filters

Sample Concentration(ng/μL) Initial_volume(μL) Yield(ng)
Filter #10 pH 7.1 5/15/17 0.296 50 14.65
Filter #7 pH 8.2 5/15/17 8.44 50 422
Filter #7 pH 8.2 5/1917 2.52 50 126
Filter #10 pH 7.1 5/22/17 2.0 50 100
Filter #10 pH 7.1 5/26/17 11.9 50 595

Samples were stored Sam gDNA Box #2, positions G8 – H3. (FTR 213, #27 (small -20oC frezer))

DNA Isolation & Quantification – Geoduck larvae metagenome filter rinses

This is another attempt to isolate DNA from two more of the geoduck hatchery metagenome samples Emma delivered on 20180313.

The previous attempt, using DNAzol, did not yield any DNA.

I isolated DNA from the following two samples:

  • MG 5/19 #4
  • MG 5/26 #4

I used the DNA Stool Kit (Qiagen), following the “Stool Human DNA” protocol with the following changes:

  • Incubated @ 95oC for 5mins after initial addition of Buffer ASL. This is a lysis step that might help increase yields (see the “Stool Pathogen Detection” protocol)
  • Did not add InhibitEX Tablet. Deemed unnecessary, since these weren’t stool samples.
  • Eluted in 50μL of Buffer AE

I opted to follow the “Stool Human DNA” protocol, as it processes a larger portion of the initial sample, compared to the “Stool Pathogen Detection” protocol (600μL vs. 200μl)

Samples were quantified using the Roberts Lab Qubit 3.0 with the Qubit High Sensitivity dsDNA Kit (Invitrogen).

10μL of each sample were used.

Results:

Neither sample yielded any detectable DNA. Will discuss with Steven.

DNA Isolation & Quantification – Geoduck larvae metagenome filter rinses

Isolated DNA from two of the geoduck hatchery metagenome samples Emma delivered on 20180313 to get an idea of what type of yields we might get from these.

  • MG 5/15 #8
  • MG 5/19 #6

As mentioned in my notebook entry upon receipt of these samples, I’m a bit skeptical will get any sort of recovery, based on sample preservation.

Isolated DNA using DNAzol (MRC, Inc.) in the following manner:

  1. Added 1mL of DNAzol to each sample; mixed by pipetting.
  2. Added 0.5mL of 100% ethanol; mixed by inversion.
  3. Pelleted DNA 5,000g x 5mins @ RT.
  4. Discarded supernatants.
  5. Wash pellets (not visible) with 1mL 75% ethanol by dribbling down side of tubes.
  6. Pelleted DNA 5,000g x 5mins @ RT.
  7. Discarded supernatants and dried pellets for 5mins.
  8. Resuspended DNA in 20uL of Buffer EB (Qiagen).

Samples were quantified using the Roberts Lab Qubit 3.0 with the Qubit High Sensitivity dsDNA Kit (Invitrogen).

5uL of each sample were used.

Results:

As expected, both samples did not yield any detectable DNA.

Will discuss with Steven on what should be done with the remaining samples.

DNA Isolation & Quantification – Crassostrea virginica Mantle gDNA

DNA was isolated from a single adult Eastern oyster (Crassostrea virginica) for a pilot project with Qiagen to test their new DNA bisulfite conversion kit. The oyster was obtained yesterday afternoon (20171210) from the Taylo rShellfish Pioneer Square location. The oyster was stored @ 4C O/N.

The oyster was shucked and four pieces of upper mantle tissue (~35mg each) were snap frozen in liquid nitrogen (LN2). Tissues were pulverized under LN2 and then DNA was isolated separately from each sample using the E.Z.N.A. Mollusc DNA Kit (Omega) according to the manufcaturer’s protocol.

Samples were eluted with 100uL of Elution Buffer and were pooled into a single tube.

The gDNA was quantified using the Qubit 3.0 (Invitrogen) and Qubit dsDNA Broad Range Kit (Invitrogen), using 5uL of sample.

Results:

Qubit (Google Sheet): 20171211_qubit_virginica_DNA

Concentration is 58.4ng/uL.

That makes the total yield ~23.36ug (23360ng). This is more than enough to perform two separate MeDIP preps and two separate reduced representation digestions with MspI.

Will proceed with shearing of DNA for MeDIP.

DNA Isolation & Quantification – C. virginica Gonad gDNA

I isolated DNA from the Crassotrea virginica gonad samples sent by Katie Lotterhos using the E.Z.N.A. Mollusc Kit with the following modifications:

  • Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
  • No optional steps were used
  • Eluted each in 100μL of Elution Buffer and pooled into a single sample

NOTE: Sample 034 did not process properly (no phase separation after 24:1 chlorform:IAA addition – along with suggested additions of ML1 Buffer) and was discarded.

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 2μL of DNA sample.

Samples were stored in the same box the tissue was delivered in and stored in the same location in our -80C: rack 8, row 5, column 4.

Results:

Qubit (Google Sheet): 20171114_qubit_Cvirginica_gDNA

Ample DNA in all samples for MBDseq. (Refer to “Original Sample Conc.” column in spreadsheet.)

Will let Steven & Katie know.

DNA Isolation – Geoduck gDNA for Illumina-initiated Sequencing Project

We were previously approached by Cindy Lawley (Illumina Market Development) for possible participation in an Illumina product development project, in which they wanted to have some geoduck tissue and DNA on-hand in case Illumina green-lighted the use of geoduck for testing out the new sequencing platform on non-model organisms. Well, guess what, Illumina has give the green light for sequencing our geoduck! However, they need at least 4μg of gDNA, so I’m isolating more.

Isolated DNA from ctenidia tissue from the same Panopea generosa individual used for the BGI sequencing efforts. Tissue was collected by Brent & Steven on 20150811.

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from five separate ~60mg pieces of ctenidia tissue according to the manufacturer’s protocol, with the following changes:

  • Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
  • Incubated homogenate at 60C for 1hr
  • No optional steps were used
  • Performed three rounds of 24:1 chloroform:IAA treatment
  • Eluted each in 50μL of Elution Buffer and pooled into a single sample

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 162ng/μL (Quant data is here [Google Sheet]: 20170105_gDNA_geoduck_qubit_quant

Yield is great (total = ~32μg).

Evaluated gDNA quality (i.e. integrity) by running 162ng (1μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O’GeneRuler DNA Ladder Mix (ThermoFisher).

 

Results:

 

 

DNA looks good: bright high molecular weight band, minimal smearing, and minimal RNA carryover (seen as more intense “smear” at ~500bp).

Will send off 10μg (they only requested 4μg) so that they have extra to work with in case they come across any issues.

DNA Isolation – Geoduck gDNA for Potential Illumina-initiated Sequencing Project

We were approached by Cindy Lawley (Illumina Market Development) yesterday to see if we’d be able to participate in some product development. We agreed and need some geoduck DNA to send them, in case she’s able to get our species greenlighted for use.

Isolated DNA from ctenidia tissue from the same Panopea generosa individual used for the BGI sequencing efforts. Tissue was collected by Brent & Steven on 20150811.

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from two separate 50mg pieces of ctenidia tissue according to the manufacturer’s protocol, with the following changes:

  • Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
  • Incubated homogenate at 60C for 1hr
  • No optional steps were used
  • Performed three rounds of 24:1 chloroform:IAA treatment
  • Eluted each in 50μL of Elution Buffer and pooled into a single sample

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 19.4ng/μL (Quant data is here [Google Sheet]: 20161221_gDNA_qubit_quant

Yield is low (~1.8μg), but have enough to satisfy the minimum of 1μg requested by Cindy Lawley.

Evaluated gDNA quality (i.e. integrity) by running ~250ng (12.5μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O’GeneRuler DNA Ladder Mix (ThermoFisher).

 

Results:

 

 

 

 

Overall, the sample looks good. Strong, high molecular weight band is present with minimal smearing. However, there is a smear in the ~500bp range. This is most likely residual RNA. This is surprsing since the E.Z.N.A Mollusc Kit includes n RNase step. Regardless, having intact, high molecular weight DNA is the important part for this project. Will prepare to send remainder (~1.5μg) of geoduck to Illumina with other requested samples.

DNA Isolation – Ostrea lurida DNA for PacBio Sequencing

In an attempt to improve upon the partial genome assembly we received from BGI, we will be sending DNA to the UW PacBio core facility for additional sequencing.

Isolated DNA from mantle tissue from the same Ostrea lurida individual used for the BGI sequencing efforts. Tissue was collected by Brent & Steven on 20150812.

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from two separate 50mg pieces of mantle tissue according to the manufacturer’s protocol, with the following changes:

  • Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
  • Incubated homogenate at 60C for 1.5hrs
  • No optional steps were used
  • Performed three rounds of 24:1 chloroform:IAA treatment
  • Eluted each in 50μL of Elution Buffer and pooled into a single sample

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 326ng/μL (Quant data is here [Google Sheet]: 20161214_gDNA_Olurida_qubit_quant

Yield is good and we have more than enough (~5μg is required for sequencing) to proceed with sequencing.

Evaluated gDNA quality (i.e. integrity) by running ~500ng (1.5μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O’GeneRuler DNA Ladder Mix (ThermoFisher).

Results:

 

 

Overall, the gel looks OK. A fair amount of smearing, but a strong, high molecular weight band is present. The intensity of the smearing is likely due to the fact that the gel is overloaded for this particular well size. If I had used a broader comb and/or loaded less DNA, the band would be more defined and the smearing would be less prominent.

Will submit sample to the UW PacBio facility tomorrow!

DNA Isolation – Oly gDNA for BS-seq

Need DNA to prep our own libraries for bisulfite-treated high-throughput sequencing (BS-seq).

Isolated gDNA from the following tissue samples stored in RNAlater (tissue was not weighed) using DNAzol:

2NF1
2NF2
2NF3
2NF4
2NF5
2NF6
2NF7
2NF8
1NF11
1NF12
1NF13
1NF14
1NF15
1NF16
1NF17
1NF18

The sample coding breaks down as follows (see the project wiki for a full explanation):

2NF#

2 = Oysters outplanted in Fidalgo Bay

NF = Broodstock originated in Fidalgo Bay

# = Sample number

1NF#

1 = Oysters outplanted in Oyster Bay

NF = Broodstock originated in Fidalgo Bay

# = Sample number

 

DNA was isolated in the following manner:

  • Homogenized tissues in 500μL of DNAzol (Molecular Research Center; MRC).
  • Added additional 500μL of DNAzol.
  • Added 10μL of RNase A (10mg/mL, ThermoFisher); incubated 10mins @ RT.
  • Added 300μL of chloroform and mixed moderately fast by hand.
  • Incubated 5mins @ RT.
  • Centrifuged 12,000g, 10mins, RT.
  • Transferred aqueous phase to clean tube.
  • Added 500μL of 100% EtOH and mixed by inversion.
  • Pelleted DNA 5,000g, 5mins @ RT.
  • Performed 3 washes w/70% EtOH.
  • Dried pellet 3mins.
  • Resuspended in 100μL of Buffer EB (Qiagen).
  • Centrifuged 12,000g, 10mins, RT to pellet insoluble material.
  • Transferred supe to clean tube.

The samples were quantified using the Qubit dsDNA BR reagents (Invitrogen) according to the manufacturer’s protocol and used 1μL of sample for measurement.

Results:

Qubit data (Google Sheet): 20151216_Oly_gDNA_qubit_quants

SAMPLE CONCENTRATION (ng/μL)
2NF1 76.4
2NF2 175
2NF3 690
2NF4 11.7
2NF5 142
2NF6 244
2NF7 25
2NF8 456
1NF11 182
1NF12 432
1NF13 155
1NF14 21
1NF15 244
1NF16 112
1NF17 25.2
1NF18 278

 

Will run samples on gel tomorrow to evaluate gDNA integrity.

DNA Isolation – Olympia Oyster Outer Mantle gDNA

Isolated additional gDNA for the genome sequencing. To try to improve the quality (260/280 & 260/230 ratios) of the gDNA, I added a chloroform step after the initial tissue homogenization.

Used 123mg of Ostrea lurida outer mantle collected by Brent & Steven on 20150812.

  • Homogenized in 500μL of DNAzol.
  • Added additional 500μL of DNAzol.
  • Centrifuged 12,000g, 10mins, @ RT.
  • Split supernatant equally into two tubes.
  • Added 500μL of chloroform and mixed moderately fast by hand.
  • Centrifuged 12,000g, 10mins, RT.
  • Combined aqueous phases from both tubes in a clean tube.
  • Added 500μL of 100% EtOH and mixed by inversion.
  • Spooled precipitated gDNA and transferred to clean tube.
  • Performed 3 washes w/70% EtOH.
  • Dried pellet 3mins.
  • Resuspended in 200μL of Buffer EB (Qiagen).
  • Centrifuged 10,000g, 5mins, RT to pellet insoluble material.
  • Transferred supe to clean tube.

DNA was quantified using two methods: NanoDrop1000 & Qubit 3.0 (ThermoFisher).

For the Qubit, the samples were quantified using the Qubit dsDNA BR reagents (Invitrogen) according to the manufacturer’s protocol and used 1μL of sample for measurement.

Results:

Qubit Data (Google Sheet): 20151125_qubit_gDNA_geoduck_oly_quants

METHOD CONCENTRATION (ng/μL) TOTAL (μg)
Qubit 137 27.4
NanoDrop1000 295 59.0

 

Yield is solid. We should finally have sufficient quantities of gDNA to allow for BGI to proceed with the rest of the genome sequencing! Will run sample on gel to evaluate integrity and then send off to BGI.

The NanoDrop & Qubit numbers still aren’t close (as expected).

The addition of the chloroform step definitely helped improve the 260/280 OD ratio (see below). However, the addition of that step had no noticeable impact on the 260/230 OD ratios, which is a bit disappointing.

 

NanoDrop Absorbance Values & Plots