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 Extractions Comparisons – Test Water Filters

Trying out the following modifications to the filters to potentially improve extraction efficiency:

  1. 3rds (three tubes per filter; labeled as C0, C10, C100)

Each tube received twice the recommended volume of Buffer AL (400uL) and twice the recommend volume of Proteinase K. Samples were vortexed for ~30s and then incubated O/N @ 56C. Same samples were then combined into a single tube and passed through a Qiagen column. Samples were then processed according to the manufacturer’s protocol and eluted with 100uL of Buffer AE. Samples were then spec’d on the Teacan plate reader, along with the test water filter extractions from 20111212, using 100uL Pico Green solution (a 1:200 dilution of stock Pico Green in 1x Low TE Buffer) in each well and 1uL of template in each well.

Samples were then stored in Sam’s 4C Box.

Results:

Raw fluorescence readings, plate layout, and sample concentrations are here.

Standard Curve

y = 1807.1x + 2428.3

d = 98.525

r = 0.99997

Quick summary of results: No method appears to better than any of the others, as they all have very similar concentrations at each respective dilution. However, cutting the filter into halves is fastest when it comes to processing. Will qPCR to assess if there are noticeable differences between extraction methods at the molecular level.

DNA Extractions Comparison – Test Water Filters

Trying out the following modifications to the filters to potentially improve extraction efficiency:

  1. Strips (single tube per filter; labeled as A0, A10, A100)

  2. Halves (two tubes per filter; labeled as B0, B10, B100)

All tubes were 1.5mL snap cap tubes. Each tube received twice the recommended volume of Buffer AL (400uL) and twice the recommend volume of Proteinase K. Samples were vortexed for ~30s and then incubated O/N @ 56C. Same samples were then combined into a single tube and passed through a Qiagen column. Samples were then processed according to the manufacturer’s protocol and eluted with 100uL of Buffer AE.

Samples were then stored in Sam’s 4C Box. Samples will be quantified once I perform a third extraction method.

DNA Extractions – Water Filters from Summer 2010

Extracted DNA from water filters collected during Summer 2010 from various locations using the DNeasy Kit. Samples were eluted with 100uL of Buffer AE and stored @ 4C. Spreadsheet indicating which samples were extracted is here. Samples were quantified on 20111129 using 1uL of template, in 100uL of PicoGreen solution prepared in Low TE. After a few tries, a “low curve” was required to be used to accurately quantify the DNA in these samples.

Results:

y = 4010.4x + 4324.8

d = 115.35

r = 0.99997

Plate layout and average concentration of samples is here.