TITLE: DNA extraction protocol for animal blood samples using the E.Z.N.A blood mini kit
AUTHORS: Louis-Stéphane Le Clercq, Desiré Lee Dalton, Antoinette Kotzé, Paul Grobler

[DESCRIPTION]
The E.Z.N.A. Blood DNA Mini Kit provides an easy and rapid method for the isolation of genomic DNA for consistent PCR and Southern analysis. Up to 250 μL fresh, frozen, or anticoagulated whole blood can be readily processed at one time. The E.Z.N.A. Blood DNA Mini Kit can also be used for the preparation of genomic DNA from buffy coat, serum, plasma, saliva, buccal swabs, and other body fluids. The E.Z.N.A. Blood DNA Kit allows for single or multiple simultaneous processing of multiple samples. There is no need for phenol/chloroform extractions, and time-consuming steps are eliminated (e.g. precipitation using isopropanol or ethanol). Purified DNA obtained with the E.Z.N.A. Blood DNA Kit is ready for applications such as PCR, restriction digestion, and Southern blotting.

[BEFORE_START]
Prepare HBC Buffer and DNA Wash Buffer according to the directions.
Set water bath, incubator, or heat block to 65°C.
Heat the Elution Buffer to 65°C.

[GUIDELINES]
Read and review the included product manual before starting.

[STEPS]
SECTION: Lyse
1. Transfer the    into a sterile microcentrifuge tube and bring the volume up to 250 µL with 10mMTris-HCl, PBS, or Elution Buffer (provided).

SECTION: Adjust binding conditions
5. Add 260 µL   . Vortex at maximum speed for 20 s  seconds.

SECTION: Bind
7. Insert a HiBind DNA Mini Column (provided) into a 2 mL Collection Tube.

  

SECTION: Wash
14. Add 700 µL DNA Wash Buffer (provided).

 

SECTION: Dry
18. Centrifuge the empty HiBind DNA Mini Column at 13000 x g, 2 min, 21 °C .
 


SECTION: Elute
19. Transfer the HiBind DNA Mini Column into a nuclease-free 2 mL microcentrifuge tube.
SECTION: Lyse
2. Add 25 µL Proteinase K Solution (provided), 250 µL BL Buffer (provided), and 5 µL     (50 mg/mL). 

SECTION: Lyse
3. Vortex at maximum speed for 15 s seconds.

SECTION: Lyse
4. Incubate at 65 °C for 10 min minutes. Vortex briefly once during incubation.

  

SECTION: Adjust binding conditions
6. Centrifuge briefly to collect any drops from the inside of the lid. 

SECTION: Bind
8. Transfer the entire sample to the column.
SECTION: Bind
9. Centrifuge at 11000 x g, 1 min, 21 °C 

SECTION: Bind
11. Add 500 µL HBC Buffer.
 


SECTION: Bind
10. Discard the filtrate and the Collection Tube. Insert the HiBind DNA Mini Column into a new 2 mL Collection Tube. 
SECTION: Bind
12. Centrifuge at 11000 x g, 1 min, 21 °C 

SECTION: Bind
13. Discard the filtrate and reuse Collection Tube. 
SECTION: Wash
15. Centrifuge at 10000 x g, 1 min, 21 °C 

SECTION: Wash
16. Discard the filtrate and reuse the Collection Tube.
SECTION: Wash
17.   

SECTION: Elute
20. Add 50 µL  Elution Buffer heated to 65 °C .

SECTION: Elute
21.  Incubate the HiBind DNA Mini Column at 65 °C for 5 min .

SECTION: Elute
22. Centrifuge at 13000 x g, 2 min, 21 °C .

SECTION: Elute
23. Optional: Apply filtrate to column and repeat centrifugation.
SECTION: Store
24. Store eluted DNA at -20 °C .

Samples extracted using this protocol were submitted to NCBI BioSample and linked to BioProject PRJNA737185.