Tag Archives: DNeasy

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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DNA Extraction – Taylor Water Filter Samples from 2011

Extracted DNA from the following water filter samples using the Qiagen DNeasy Blood & Tissue Kit:

  • 158
  • 200
  • 279
  • 313
  • 341
  • 410
  • 433
  • 503
  • 551
  • 604

Filters were cut into ~13 pieces and placed in 1.5mL snap cap tubes containing 50uL of Proteinase K and 400uL of Buffer AL. Samples were incubated O/N @ 56C. Tubes were spun @ 16,000g @ RT for 2mins. 400uL of 100% EtOH was added to each tube and vortexed. Tubes were spun @ 16,000g @ RT for 2mins. Supe was transferred to Qiagen column. Qiagen protocol was followed from this point on. Samples were eluted with 100uL of Buffer AE and stored @ 4C.

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DNA Extraction – Abalone Water Filters from Summer 2010

Extracted DNA from water filters collected during Summer 2010 from various locations using the DNeasy Kit. Based on extraction methodology tests (see 20111221), filter were each cut into ~13 strips and placed in a 1.5mL snap cap tube containing 400uL of Buffer AL and 50uL of Proteinase K. The volumes of both reagents are double the kit recommendation. Samples were vortexed thoroughly and incubated 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 kit protocol was followed for the remainder of the procedure. Samples were eluted with 100uL of Buffer AE and stored @ 4C. Spreadsheet indicating which samples were extracted is here.

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