DNA extraction protocol for animal blood samples using the E.Z.N.A blood mini kit
Louis-Stéphane Le Clercq, Desiré Lee Dalton, Antoinette Kotzé, Paul Grobler
Abstract
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.
Steps
Lyse
Transfer the Sample into a sterile microcentrifuge tube and bring the volume up to 250µL with 10mMTris-HCl, PBS, or Elution Buffer (provided).
Add 25µL Proteinase K Solution (provided), 250µL BL Buffer (provided), and 5µL
Vortex at maximum speed for 0h 0m 15s seconds.
Incubate at 65°C for 0h 10m 0s minutes. Vortex briefly once during incubation.
Adjust binding conditions
Add 260µL 0h 0m 20s seconds.
Centrifuge briefly to collect any drops from the inside of the lid.
Bind
Insert a HiBind DNA Mini Column (provided) into a 2 mL Collection Tube.
Transfer the entire sample to the column.
Centrifuge at 11000x g,21°C
Discard the filtrate and the Collection Tube. Insert the HiBind DNA Mini Column into a new 2 mL Collection Tube.
Add 500µL HBC Buffer.
Centrifuge at 11000x g,21°C
Discard the filtrate and reuse Collection Tube.
Wash
Add 700µL DNA Wash Buffer (provided).
Centrifuge at 10000x g,21°C
Discard the filtrate and reuse the Collection Tube.
Dry
Centrifuge the empty HiBind DNA Mini Column at 13000x g,21°C .
Elute
Transfer the HiBind DNA Mini Column into a nuclease-free 2 mL microcentrifuge tube.
Add 50µL Elution Buffer heated to 65°C .
Incubate the HiBind DNA Mini Column at 65°C for 0h 5m 0s .
Centrifuge at 13000x g,21°C .
Optional: Apply filtrate to column and repeat centrifugation.
Store
Store eluted DNA at -20°C .
Samples extracted using this protocol were submitted to NCBI BioSample and linked to BioProject PRJNA737185.
