Tag Archives: DNeasy

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 – Metagenomics Water Filters

Isolated DNA from the following two 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 10μL of each sample for measurement (see Results for update).

Results:

Raw data (Google Sheet): 20180411_qubit_metagenomics_filters

Sample Concentration(ng/μL) Initial_volume(μL) Yield(ng)
filter 5/22 #7 pH8.2 20.8 50 1040
filter 5/26 #7 pH8.2 11.6 50 580

NOTE: For “filter 5/22 #7 pH8.2″ the initial quantification using 10μL ended up being too concentrated. Re-ran using 5μL.

Both samples have yielded DNA. This is, obviously, an improvement over the previous attempts to isolate DNA from ammonium bicarbonate filter rinses that Emma supplied me with.

Will discuss with Steven and get an idea of which filters to isolate additional DNA from.

Samples were stored Sam gDNA Box #2, positions G6 & G7. (FTR 213, #27 (small -20oC frezer)

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DNA Extraction & Quantification- Ava Withering Syndrome Transmission Study Water Filters

DNA was extracted from filters using the Qiagen DNeasy Blood & Tissue Kit (spin column protocol). Filters were incubated in 400uL of Buffer AL (twice the volume in the Qiagen protocol in order to completely coat the filters) and 50uL of Proteinase K (twice the volume in the Qiagen protocol) at 56C O/N. After incubation, 400uL (twice the volume in the Qiagen protocol) of 100% EtOH was added to each tube and vortexed thoroughly. The supernatant was transferred to the Qiagen spin columns and the Qiagen protocol was followed. Samples were eluted with 100uL of Buffer AE.

After extraction, the samples were quantified using the Roberts Lab Qubit 3.0 (Life Technologies) using the Qubit dsDNA HS reagents. Used 1μL of each sample.

The list of samples are listed below.

Results:

Concentrations are very low for both samples. This may, or may not, be expected, depending on volume of water filtered, where it was collected from, etc.

Raw Qubit Readout (Google Sheet): 20160818_DNA_quant_Qubit_Ava_abalone_WS
Master spreadsheet for these, and future, samples for this project (Google Sheet): Ava WS Transmission DNA Extractions

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DNA Isolation – Olympia Oyster Populations for RAD Sequencing

Olympia oysters from three different Puget Sound locations/populations (HL, NF, and SN) were collected and stored @ -80C on 8/29/2103.

We removed whole bodies from 32 oysters (randomly selected; ~5 -10mm “diameter” shells) from each population and placed them into a 96 well DNeasy Blood & Tissue Kit (Qiagen). DNA was prepared/isolated according to the manufacturer’s protocol.

DNA was eluted once with 200uL of Buffer AE and stored @ 4C.

Plate is called: Oly Oyster gDNA-01

Plate layout can be found here: 20141022-OlyRADdnaConcentrations

UPDATE 20141017

Steven ran the samples out on a gel for quality assessment. His notebook entry can be seen here:

Gel layout info and image of gel 1 of 2: http://sr320.tumblr.com/post/100245499294

Images of gel 2 of 2: http://sr320.tumblr.com/post/100231194034

The samples are all heavily smeared, suggesting heavy degradation. Will compare Qiagen kit with DNazol on some of the samples from 8/29/2013, as well as samples more recently collected/frozen.

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DNA gel – Claire’s C.gigas Female Gonad and Mac’s C.gigas Gonad

Ran out 2uL of Clair’es C.gigas female gonad gDNA (from 20140328) and Mac’s C.gigas gonad gDNA (from 20140402) for quality assessment. Both samples had been isolated using Qiagen’s Blood & Tissue DNeasy Kit. 2uL of each sample was run on a 0.8% 1x TBE gel.

Results:

Loading:

Lane 1 – Hyperladder 1 (Bioline)

Lane 2 – Claire’s gDNA

Lane 3 – mac’s gDNA

Both samples show an extremely high amount of smearing. Additionally, both samples have definitive bands that correspond to ~1300bp and ~850bp.

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DNA Isolation – Mackenzie’s C.gigas Gonad Sample

Mac’s been having some difficulty getting good quality gDNA from some of her gonad samples, so she asked me to give it a shot.

Isolated DNA from 10mg (0.010g) of C.gigas gonad tissue using the Qiagen Blood & Tissue DNeasy Kit, with the following changes:

  1. Incubated sample in Buffer ATL + Proteinase K @ 56C for 3hrs

  2. Eluted sample in 100uL of Buffer AE.

Spec’d on NanoDrop1000.

Results:

DNA looks good, both 260/280 ratio and yield. The 260/230 ratio isn’t perfect, but it’s much better than what Mac was seeing. After showing her this, she’s decided to have me isolate DNA from the rest of her samples.

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DNA Isolation – C.gigas Female Gonads (from frozen)

Isolated gDNA from Claire’s “Female DNA” (from 05/16/2013) using the Qiagen Blood & Tissue DNeasy Kit according to the manufacturer’s protocol, with the following changes:

  1. Incubated sample in Buffer ATL + Proteinase K @ 56C for 3hrs. Vortexed once each hour.

  2. Eluted with 100uL of Buffer AE.

Results:

Excellent yield and quality is good, although both the 260/280 and 260/230 ratios are on the high side. However, these high values could be an artifact of the high sample concentration (this is a common “issue” with the NanoDrop).

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DNA Isolation – Geoduck

Isolated additional geoduck gDNA from the two fresh (now frozen) geoduck’s that Brent provided me with on 20140212 so that we can potentially isolate RNA from the same geoducks to tie in with the DNA Illumina sequencing that Axa will be conducting. Isolated DNA using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol (incubated minced siphon tissue at 56C for 3hrs). Eluted with 75uL of ddH2O and spec’d on NanoDrop1000.

Note: Initial specs were too low for Axa’s requirements (50uL, >= 500ng/uL). SpeedVac’d samples to concentrate, brought volume to 55uL and then spec’d on NanoDrop1000.

Results:

Samples look good. Will send Axa 50uL of all samples, excluding GD01 since that sample is below his desired concentration AND I believe he probably doesn’t want to wait for this DNA any longer.

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DNA Isolation – Geoduck

Since yesterday’s DNA isolation failed to yield sufficient quantity of DNA from the ethanol-fixed samples, I isolated additional DNA from the same samples.

Isolated gDNA from 6 geoduck siphon samples provided by Brent using the Qiagen DNeasy Spin Kit. Samples were as follows:

  • Langley 006
  • Langley 007
  • Langley 008
  • Langley 009

The Langley samples were fixed in ethanol in 2006. Geoduck 01/02 were fresh geoduck siphon samples, taken from two live, juvenile geoduck (the rest of the bodies were frozen at -80C).

The samples were processed according to the Qiagen protocol (but utilized an overnight incubation at 56C with Proteinase K), eluted in 50uL of Buffer AE, combined with the corresponding DNA from yesterday, mixed throughly and spec’d on a NanoDrop1000.

Results:

Now have sufficient quantity of DNA for all four of these samples. Will contact Axa (the person who this DNA is intended for) to see if he requires a specific concentration/volume.

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DNA Isolation – Geoduck

Isolated gDNA from 6 geoduck siphon samples provided by Brent using the Qiagen DNeasy Spin Kit. Samples were as follows:

  • Langley 006
  • Langley 007
  • Langley 008
  • Langley 009
  • Geoduck 01
  • Geoduck 02

The Langley samples were fixed in ethanol in 2006. Geoduck 01/02 were fresh geoduck siphon samples, taken from two live, juvenile geoduck (the rest of the bodies were frozen at -80C).

The samples were processed according to the Qiagen protocol (but utilized an overnight incubation at 56C with Proteinase K), eluted in 100uL of Buffer AE and spec’d on a NanoDrop1000.

Results:

The person who needs these samples (Axa) needs at least 25ug of DNA. The two fresh samples (Geoduck 01 and Geoduck 02) yielded more than sufficient quantities of DNA. The Langley (i.e. ethanol-fixed) samples did not yield sufficient DNA and I will need to isolate additional DNA from these samples.

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