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    Protocol For Isolation of DNA From Salvia

    互联网

    518

    实验原理

     

    E.Z.N.A.® Mag-Bind Forensic DNA Isolation Kits use the reversible binding properties of the Mag-Bind® paramagnetic particles to provide a fast and flexible method for isolating genomic DNA from different forensic sources. Samples are first lysed with a specially formulated buffer containing detergent in the presence of Proteinase K. After adjust the binding condition, the sample was mixed with Mag-Bind particles and the genomic DNA was bound to the surface of Mag-Bind magnetic particles. Proteins, polysaccharides, and cellular debris are efficiently washed away with few wash steps. Pure DNA is then eluted in water or low ionic strength buffer. Purified DNA can be directly used in downstream applications without the need for further purification.

    实验步骤

     

    1. Collect 200ul saliva in a 1.5 mL centrifuge tube contains 200ul Buffer MSL and 20ul of Proteinase K.

    2. Mix the sample throughly by vortexing or pipetting up and down for 20 times.

    3. Incubate at 65°C for 30 minutes.

    4. Centrifuge at 14,000 x g for 2 minutes and transfer the sample to a new 1.5 ml tube.

    5. Optional: If RNA-free DNA is desired, add 10 μl of RNase A (25mg/ml) and incubate at room temperature for 5 minutes.

    6. Add 10μl Mag-Bind Particles followed by 290 μl of absolute ethanol. Mix throughly by vortexing or pipetting up and down for 20 times.

    7. Incubate at room temperature for 5 minutes.

    8. Place the tube or plate to a magnetic separation device suitable for 1.5 ml tube or 96-well microplate to magnetize the Mag-Bind particles. The solution should be cleared after all the magnetic beads are pelleted.

    9. Carefully remove and discard the cleared supernatant by pipetting.

    10. Remove the tube or plate containing the Mag-Bind particles from the magnetic separation device.

    11. Add 300ul MP Buffer to each sample and mix throughly by vortexing or pipetting up and down for 20 times.

    12. Carefully remove and discard the cleared supernatant by pipetting.

    13. Remove the tube or plate containing the Mag-Bind particles from the magnetic separation device.

    14. Add 500 ul or 300 ul SPM Buffer to each sample and mix throughly by vortexing or pipetting up and down for 20 times.

    Note: SPM Buffer must be diluted with ethanol before use.

    15. Place the tube or plate to a magnetic separation device suitable for 1.5 ml tube or 96-well microplate to magnetize the Mag-Bind particles.

    16. Carefully remove and discard the cleared supernatant by pipetting.

    17. Remove the tube or plate containing the Mag-Bind particles from the magnetic separation device.

    18. Wash the Mag-Bind particles again by repeating step 15-17 with SPM Buffer.

    19. Leave the tube to air dry on the magnetic separation device for 5-10 minutes. Remove any residue liquid from tube by pipetting.

    20. Remove the tube or plate containing the Mag-Bind particles from the magnetic separation device.

    21. Add 50-200ul of Elution Buffer to the tube or each well of the microplate. Mix throughly by vortexing or pipetting up and down for 50 times.

    22. Incubate at 60°C for 15 minutes.

    23. Place the tube or plate to a magnetic separation device suitable for 1.5 ml tube or 96-well microplate to magnetize the Mag-Bind particles.

    24. Carefully transfer the cleared supernatant contains eluted DNA to a clean 1.5 ml tube or microplate.

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