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))

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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.

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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.

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DNA Isolation & Quantification – Pinto Abalone

Isolated DNA from the following pinto abalone (Haliotis kamtschatkana) digestive gland tissues (stored in ethanol), collected by Sean Bennett as part of his Capstone project:

Accession Weight(mg)
15:30-01   194
15:30-04   67
15:31-01   34
15:31-02   107
15:31-03   83
15:31-04   80

Tissue was weighed and then DNA extracted.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

Used the Roberts Lab Qubit 3.0 and the Qubit hsDNA Kit (high sensitivity). Used 1uL of template for all samples.

Samples were stored at -20C in FSH240 in the “Pinto Transcriptome DNA” box.

Results:

All samples have DNA.

Concentrations (Google Sheet): 20171226_qubit_DNA_pinto_ab

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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.

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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.

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DNA Isolation – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 17 red abalone digestive gland tissue samples.

Tissue was weighed, minced with a razor blade, and transferred to 2mL snap cap tube containing 1mL of InhibtEX Buffer.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

Minced tissue was incubated at 70C O/N.

These samples were actually incubated O/N on 20171012. I dropped the rack containing these tubes after the initial incubation and these tubes popped open and spilled.

Since I used all of the tissue, I have nothing to go back to. I’ve attempted to recover as much of the remaining supernatant in each of these tubes. I brought the volume of each tube up to 600mL with Inhibitex Buffer and proceeded with the isolations.

Followed “human DNA analysis” protocol (to maximize sample recovery)
Eluted DNA with 100μL Buffer ATE
Sample information is in this spreadsheet (Google Sheet): ava_abalone_master_extraction_list

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DNA Isolation – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 96 red abalone digestive gland tissue samples.

Tissue was weighed, minced with a razor blade, and transferred to 2mL snap cap tube containing 1mL of InhibtEX Buffer.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

Minced tissue was incubated at 70C O/N
Followed “human DNA analysis” protocol (to maximize sample recovery)
Eluted DNA with 100μL Buffer ATE
Sample information is in this spreadsheet (Google Sheet): ava_abalone_master_extraction_list

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DNA Isolation – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 144 red abalone digestive gland tissue samples.

Tissue was weighed, minced with a razor blade, and transferred to 2mL snap cap tube containing 1mL of InhibtEX Buffer.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

Minced tissue was incubated at 70C O/N
Followed “human DNA analysis” protocol (to maximize sample recovery)
Eluted DNA with 100μL Buffer ATE
Sample information is in this spreadsheet (Google Sheet): ava_abalone_master_extraction_list

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DNA Isolation – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 58 red abalone digestive gland tissue samples.

Tissue was weighed, minced with a razor blade, and transferred to 2mL snap cap tube containing 1mL of InhibtEX Buffer.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Minced tissue was incubated at 70C O/N
  • Followed “human DNA analysis” protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

Sample information is in this spreadsheet (Google Sheet): ava_abalone_master_extraction_list

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