Tag Archives: DNeasy

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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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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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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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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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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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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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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gDNA Isolation

Isolated gDNA from gray whale skin, human cheek cells (my own!) and two different species of algae (species 1, species 2) using Qiagen’s DNEasy Blood & Tissue Kit according to protocol. Incubated all samples at 55C for 1hr. Eluted DNA with 50uL of Buffer AE. Spec’d samples on NanoDrop 1000.

Cheek cells were scraped from the inside of my cheek with a sterile toothpick. The toothpick was transferred to a 1.5mL snap cap tube containing the appropriate buffer. The tube was vortexed to help dislodge cells from the toothpick. The toothpick was then removed and the sample treated according to protocol.

1mL of algae cells were collected from each liquid culture, cells were pelleted by spinning 16,000g for 1min @ RT, supe removed, 180uL of Buffer ATL added and then vortexed to dislodge/break up pellet. Sample was treated according to protocol.

Results:

Well, no detectable quantities of DNA in 3 of the 4 samples. There appears to be something in the gray whale skin gDNA extraction, however the OD260/280 ratio is just crazy, leading me to believe that there’s not really any usable DNA present. Will give gray whale gDNA sample to Caroline for class and will talk with Steven and Caroline concerning their interest in performing another quick extraction on more algae to use for class this afternoon.

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gDNA Isolation

Isolated gDNA from crab (unknown species), starfish exposed to RoundUp (unknown species) and gray whale blubber using Qiagen’s DNEasy Kit, according to manufacturer’s protocol. Tissue was incubated at 55C with Proteinase K for 1hr. gDNA was eluted with 100uL of Buffer AE and spec’d.

Results:

Gray whale sample has virtually no DNA. Will try to precipitate the whale gDNA in order to obtain a more concentrated sample. The other two samples look good, with good yields and good OD260/280 ratios.

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