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

DNA Isolation – Ireland Clam Sample S/6/14 #19

Previous attempts at isolating usable DNA failed.  Previously used both the QIAamp Fast DNA Stool Kit and DNeasy Kit (Qiagen) and both yielded nothing. In a last ditch effort, since there’s no tissue left, I pellet the remaining tissue/debris, removed the EtOH and processed the sample with the DNeasy Kit (Qiagen), following the animal tissues spin protocol.  DNA was eluted with 100uL of Buffer AE.

Will quantify and PCR next week.

Phage Extraction – Withering Syndrome Phage Host gDNA Digestion and Capsid Digestion

DNased the phage extracted by Lisa on unknown date. Lisa resuspended the phage in TM buffer (see Fujii et al. – 2004 – Isolation and characterization of the bacteriophage WO from Wolbachia, an arthropod endosymbiont), with a resulting volume of 82uL. Sample was DNased with 2U (2uL) DNase I (Thermo Scientific) and incubated @ RT for 15mins.

Sample was then treated with lysis buffer (final concentrations: EDTA 20uM, SDS 0.5%, Proteinase K [Fermentas] 50ug/mL). Final volume of reaction was brought up to 200uL with TM buffer and incubated @ 60C for 1hr.

Sample volume was brought up to 220uL with TM buffer, then processed with a DNEasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol. Sample was eluted 2 x 100uL of Buffer AE and stored @ 4C. Did not spec due to expected low quantity of DNA; did not want to waste.

Next up is cloning and sequencing the phage. Will likely digest with EcoRI and clone into pUC19 (taken from the TOPO TA cloning kit).

DNA Isolation – “Big Reds” Tank Water Filters

In an attempt to get a water sample that has higher copy numbers for use in the withering syndrome qPCR assay validation procedures, I isolated gDNA from two water filters from the “big reds” abalone tank. Water filters were numbered 4 and 5, with no other information.

Filters were cut into ~13 strips and processed using the Qiagen DNeasy Blood & Tissue Kit. The filters were mixed with 400uL of Buffer AL and 40uL of Proteinase K (both volumes are double what is recommended in the Qiagen protocol). Samples were vortexed and incubated O/N at 56C. After incubation, 400uL of 100% EtOH was added to each sample (this is double the volume recommended by Qiagen). The manufacturer’s protocol was followed for the remainder. Samples were eluted with 100uL of Buffer AE.

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.

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.

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.

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.

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.

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.