TITLE: DNA Extraction from Swabbed Feces for Depositor Species Identification - 2024
AUTHORS: Karla Vargas

[DESCRIPTION]
Protocol developed by the Culver Lab at the University of Arizona to extract DNA from feces for depositor species identification

[STEPS]
SECTION: DAY 1
2. Saturate cotton-tipped applicators with PBS buffer.
● Keep cotton-tipped applicators in PBS buffer for at least 3 minutes.
SECTION: DAY 1
3. Pipette 25 μl of proteinase K and 300 μl of ATL buffer (from DNeasy kit) into a new, labeled 2 ml microcentrifuge tube.
SECTION: DAY 1
4. Place a sheet of copy paper on a stainless-steel tray to analyze scat samples for swabbing. Swab the surface of the scat with PBS-saturated applicator. Cut the applicator tip and place it in the 2 ml microcentrifuge tube from step 2.
●  Bleach and dry the tray and change the copy paper after each sample is processed. 
●  Change gloves after each scat sample is processed.
●  Scissors used to cut the applicator should be submerged in 10% bleach, rinsed, and wiped dry before cutting the next applicator.
SECTION: DAY 1
5. Incubate samples at 56°C for 24 – 48 hours.
● To reduce the risk of evaporation due to faulty caps, cover the cap of tubes with parafilm.
SECTION: DAY 2
6. Remove parafilm and spin tubes for 15 seconds to remove condensation inside the tube lids.  
SECTION: DAY 2
7. Remove the applicator and add 366 μl of AL buffer. Vortex and incubate at 56°C for 1 hour.
●  Scissors or tweezers used to remove the applicator should be submerged in 10% bleach, rinsed, and wiped dry before removing the next applicator.
SECTION: DAY 2
8. Add 366 μl of 100% ethanol and mix by inverting. Continue to step 8 or store at 4°C (stable for weeks).
SECTION: DAY 2
9. Label spin columns provided by Qiagen kit and carefully apply 500 μl of lysate from step 7 to the column without moistening the rim.
SECTION: DAY 2
10. Centrifuge according to Qiagen DNeasy protocol >= 6000 x g (8000 rpm) for 1 min.  
SECTION: DAY 2
11. Place the columns in a new 2 ml collection tube, and discard the tube containing the filtrate. 
SECTION: DAY 2
12. Carefully open the column, apply a second aliquot of 500 μl of lysate and centrifuge according to Qiagen DNeasy protocol >= 6000 x g (8000 rpm) for 1 min.  
SECTION: DAY 2
13. Place the columns in a new 2 ml collection tube and discard the tube containing the filtrate. 
SECTION: DAY 2
14. Prepare AE buffer: Bring Buffer AE to 70°C with the heat block. Pipette 100 ul (or 200 for less concentrated DNA) of buffer AE per sample into a new tube and place it in heat block (To use at the end)
SECTION: DAY 2
15. Add 500 μl of AW1 wash buffer to column. Centrifuge at >= 6000 x g (8000 rpm) for 1 min. Place the column in a new 2 ml collection tube. Discard the tube containing the filtrate.  
SECTION: DAY 2
16. Add 500 μl of AW2 wash buffer to column. Centrifuge at 20,000 x g (14,000 rpm) for 3 min. Place the column in a new 2 ml collection tube. Discard the tube containing the filtrate.  
SECTION: DAY 2
17. Place the columns in a new 2 ml collection tube. Centrifuge at 20,000 x g (14,000 rpm) for 1 min. (Dry spin: this step helps to eliminate the chance of possible buffer AW2 carryover). Discard the tube containing the filtrate.  
SECTION: DAY 2
18. Remove the column and place it into a labeled 1.5 ml tube. Cut off the caps.
SECTION: DAY 2
19. Add 100 - 200  μl of AE elution buffer directly to the column and incubate at room temperature for 1 to 3 minutes.
● Elution with 100 µl (instead of 200 µl) increases the final DNA concentration in the eluate
SECTION: DAY 2
20. Centrifuge columns at ≥ 6000 x g (8000 rpm) for 1 minute to elute DNA.
SECTION: DAY 2
21. Transfer DNA into labeled 0.5 ml or 1.5 ml tubes.
SECTION: DAY 2
22. Discard columns and store DNA at 4°C or -20°C for long term.
SECTION: DAY 2
23. Disinfect countertops and equipment with 10% bleach solution.

SECTION: Preparation
1. Qiagen Kit: Qiagen DNeasy Blood and Tissue extraction Mini kit 
Lab Equipment: Heat block, Vortex, Centrifuge, Pipettes, Stainless steel tray, Stainless steel Scissors, Stainless steel tweezers
Solutions and Reagents: 10x PBS (to make 1000 ml of 10X PBS: 5.56 g NaH2PO4, 23.0 g NaHPO4, 87.68 g NaCl, pH to 7.1), proteinase K, ATL buffer, AL buffer, AW1 buffer, AW2 buffer, AE buffer (provided by Qiagen kit), 100% ethanol 100% (not provided)
Consumables: 1.5 ml microcentrifuge tubes, 2 ml microcentrifuge tubes, 2 ml collection tubes, 100-1000 μl pipette tips (filter and sterile), 10-100 μl pipette tips (filter and sterile), QIAamp spin columns (provided by Qiagen kit), sterile cotton-tipped applicators 
Other supplies: Parafilm, copy paper, paper towels,10% bleach solution, gloves
Check Qiagen manual "Things to do before starting"
SECTION: DAY 2
24. Optional:
For maximum DNA yield, repeat elution once as described in steps 19-21 . This step leads to increased overall DNA yield. A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first eluate. Alternatively, to combine the eluates, the microcentrifuge tube from step 7 can be reused for the second elution step.