Extracting DNA from the first 8 of the March 2019 Sampling

Apr 4, 2019

Using Qiagen DNeasy Blood & Tissue kit and modifications from modifications from Renshaw et. al

An extraction control was included to check to see if we have any contamination in the lab. All reagents were added to the tube and it was treated like a sample except it had no filter.

Sample	Location	Sampling/Filtering Date
CGP-1	Cogshall Point	03/23/19
CGP-2	Cogshall Point	03/23/19
CGP-3	Cogshall Point	03/23/19
CGP-4	Cogshall Point	03/23/19
CS-1	Colt State Park	03/23/19
CS-2	Colt State Park	03/23/19
CS-3	Colt State Park	03/23/19
CS-4-8	Colt State Park	03/23/19
Control	none	none

sample CS-4 was from only 800mL filtered, the next extraction has the filter with the last 200mL

DNA Extraction

Filters were stored in the -80 and were removed just prior to extraction
Before extraction, the bench was wiped with 10% bleach and Type II DI water
Added to each tube with the frozen filters:
- 567µl buffer ATL
- 63µl Proteinase K
Samples were digested at 65 degrees C for 6.5 hours on the Thermomixer and shaking at 1000 rpm
After incubation, the filters were taken out of the 1.5mL tubes to get at the liquid with cleaned forceps (same as above). Excess liquid was squeezed out as best as possible with the forceps into new 5mL tubes
Original digestion tubes were spun down to pellet out any dirt and debris in the sample. The supernatant was transferred to the corresponding 5mL tube
Added to each 5mL tube and then vortexed
- 630µl buffer AL
- 630µl 100% EtOH
The sample had to run through the spin column 3 times.
- Added 600µl sample to the spin column then centrifuged at 8000rpm for 1 minute
- Transferred column to new collection tube
- Repeated above steps twice more to run all of the sample though the column
Added 500µl buffer AW1 to the columns then centrifuged at 8000rpm for 1 minute
Transferred columns to new collection tubes, added 500µl buffer AW2 and centrifuged at 14000 rpm for 3 minutes
Transferred columns to final 1.5mL tubes
- Added 50µl warmed (56 degrees C) buffer AE directly to the filter
- Incubated at room temperature for 5 minutes
- Centrifuged at 8000rpm for 1 minute
- in the same 1.5mL tube repeated above steps with 30µl warmed buffer AE
Vortexted all samples and aliquoted 30µl into separate tubes labeled 30µl for a working stock. Both sets of tubes were stored in the -20 in separate boxes

Qubit

High Sensitivity dsDNA Kit protocol [here]
All samples were read twice
All values are in ng/µl

Sample #	Standard 1	Standard 2	DNA 1	DNA 2
CGP-1	42.82	27143	56.2	56.4
CGP-2	42.82	27143	51.8	52.2
CGP-3	42.82	27143	46.4	46.2
CGP-4	42.82	27143	44.8	44.6
CS-1	42.82	27143	63.8	63.8
CS-2	42.82	27143	59.6	59.8
CS-3	42.82	27143	83.4	83.4
CS-4-8	42.82	27143	50.6	50.6
Control	42.82	27143	too low to read	-

extraction