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    Protocol For Isolation of Genomic DNA From Sperm

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    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. Add 100ul of sperm to 100ul of Buffer A in a glass (Corex) centrifuge tube. Vortex for 10 sec at full speed. Only use Corex tubes to prevent attachment of the sperm cells to the tube walls.

    2. Add 20 ul Proteinase K (20 mg/mL)and incubate for 2 hours at 60°C. Inveo rt the tube occasionally to disperse the sample or place on a rocking platform.

    3. Add 220 ul Buffer MSL to the sample and mix by vortexing.

    4. Centrifuge at full (>14,000 x g ) for 5 minutes.

    5. Transfer 400 μl sample to a new 1.5 ml tube. For 96-well microplate procedure, transfer 250μl of sample to each well of the microplate.

    6. Add 270μl (for singe tube) or 170μl absolute ethanol to each sample.

    7. Add 10ul of Mag-Bind particles and mix throughly by vortexing or pipetting up and down for 20 times.

    8. Incubate at room temperature for 5 minutes.

    9. 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.

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

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

    12. Add 500 ul (for 1.5 ml tube) or 300 ul (for 96-well plate) SPM Buffer to each sample and mix throughly by vortexing or pipetting up and down for 20 times.

    13. 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.

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

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

    16. Wash the Mag-Bind particles again with SPM Buffer by repeating step 11-13.

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

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

    19. 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.

    20. Incubate at 60°C for 15 minutes.

    21. 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.

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

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