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    Mag-Bind Blood DNA Maximum Yield Protocol (7ml-10ml)

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    实验试剂

     

    1. Absolute ethanol (96%-100%)

    实验设备

     

    1. Nuclease-free 50 ml centrifuge tube

    2. Water bath, incubator or heating block preset at 65°C

    3. Magnetic separation device for 50 ml tubes

    实验步骤

     

    1. Place two empty, uncapped 50 ml conical tubes into a 50ml tube holder.

    2. Equally divide the blood sample and transfer the sample into twp 50 ml tubes. For example: For 7ml blood, transfer 3.5ml blood into each tube.

    3. Add indicated volume of PBS, MSL, Proteinase K and RnaseA showed in the table to the sample. Mix throughly by vortexing.

    4. Incubate sample at 65°C for 20 min. Briefly vortex the tube few times during incubation. Cool the sample to room temperature by incubating at room temperature for 5 minutes.

    5. Add indicated volume of absolute ethanol showed in the table to the lysate. Mix throughly by inverting or voretxing.

    6. Add 300ul Mag-Bind Particles Solution C to the sample. Mix gently by inverting, shaking or pipetting up and down 20 times. Incubate at room temperature for 10 minutes.

    Note: Complete resuspension of cell pellet is vital for obtaining good DNA yields.

    7. Place the tubes on a magnetic separation device to magnetize the magnetic particles. Lysate will clear when the Mag-Bind particles have completely moved toward the magnet. Completely remove and discard the cleared supernatant.

    8. Remove the tubes containing the Mag-Bind particles from the magneti c separation device. Add 5 ml Buffer MP/Ethanol Mixture to the sample.

    Note: MP/Ethanol mix has to be prepared freshly.

    9. Resuspend the Mag-Bind particles pellet by vortexing. Incubate 3 minutes at room temperature. During incubation, mix the sample several times by vortexing or pipetting up and down. Complete resuspension of the Mag-Bind particles pellet by pipetting up and down or vortexing is critical to obtain good results.

    10. Transfer and combine suspended Mag-Bind Particles into a new 50 ml tube. Incubate the sample at room temperature for 5 minutes.

    11. Place the tube onto the magnetic separation device to magnetize the Mag-Bind particles. Completely remove and discard the cleared supernatant.

    12. Remove the tube containing the Mag-Bind particles from the magnetic separation device. Add 10 ml SPM Buffer to each sample.

    13. Resuspend the Mag-Bind particles pellet by vortexing. Incubate 3 minutes at room temperature. Complete resuspension of the Mag- Bind particles pellet is critical to obtain good results.

    14. Place the tube onto the magnetic separation device to magnetize the magnetic particles. Completely remove and discard the cleared supernatant

    15. Optional: Wash magnetic particles with SPM by repeating step 12-14.

    16. Leave the tube to air dry on the magnetic separation device for 5 minutes. Remove any residue liquid with a pipettor. Do not over dry the pellet.

    17. Remove the tube from the magnetic separation device. Add 2.5 ml Elution Buffer or water to elute DNA from the Mag-Bind particles. Incubate 10 minutes at 65°C. Mix throughly by vortexing for 30 seconds. Incubate 10 minutes at 65°C.

    18. Place the tube onto a magnetic separation device to magnetize the Mag-Bind particles.

    19. Transfer the cleared supernatant containing purified DNA to a new 1.5 ml tube.

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