Tag Archives: DNeasy

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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DNA Isolation – Qiagen Kit Comparison

Note: This information was added 20140407. Yes, you read that correctly.

Someone had noticed that gDNA isolated using a Qiagen DNeasy Blood & Tissue Kit we rec’d in April 2010 seemed to be yielding degraded DNA.

The two samples used for the comparison were a single tail (split in two equal weight pieces) from a juvenile salmon that was snap frozen, without preservatives, at the time of its collection. The samples were prepped. 0.5ug of eluted DNA was then run on a 1.2% agarose-TAE gel containing 0.1ug/mL of ethidium bromide. 5uL of Bioline’s Hyperladder I was loaded for size assessment (see link for marker layout).

Results:

Clearly, there’s significant quality difference! A free replacement kit was sent by Qiagen.

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gDNA Isolation – Dungan isolate MIE-14v

Cells stored in EtOH were pelleted 5000g, 10mins, 25C. A brownish smear was present along the inside of the tube after spinning; not really a pellet per se. Supe was removed and cells were washed twice with 1X PBS. The smear was reduced to a pellet after the first wash in PBS. The second wash resulted in a slightly smaller pellet, but a pellet was present nonetheless before proceeding. Cells were subjected to an enzymatic lysis in 180uL of a TE/Triton X-100/lysozyme mixture as described in the Qiagen DNeasy Kit for Gram-Positive Bacteria (and for cells having substantial cell walls). Cells were incubated in this mixture for 30mins @ 37C. 25uL of proteinase K and 200uL of Buffer AL were then added and the mixture was incubated @ 70C for 30mins. Protocol then followed the “normal” steps for isolation of gDNA. Sample was eluted in 100uL of Buffer AE and then spec’d.

Results: Well, it doesn’t look like there is any DNA at all… Will try precipitating the sample and bringing up the DNA (if there’s any) in a small volume. It should be noted that I spec’d these samples using the “RNA” setting, so the constant for concentration calcs is wrong (40), but doing quick math using the DNA constant (50) shows that there’s still almost nothing there…

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