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    Mag-Binds Plant DNA Protocol For RICE Powder or other foods

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    实验试剂

     

    Regents to be provided by user

    1. Absolute (96%-100%) ethanol

    实验设备

     

    Equipments to be provided by user

    1. Centrifuge capable of 4,000 x g.

    2. 96 well Plate

    3. Water bath preset at 65-70°C

    4. Equipment for disrupting plant tissue (MM300 Mixer Mill or Geno/Grinder 2000)

    实验步骤

     

    1. Collect 100mg rice or rice powder in a 2 ml 96 well plate with appropriate steel beads. Add 1ml Buffer SLX Minus and 4ul RNase A.

    2. Process in the mixer mill machine by following manufacturer’s instructions. Time and speed will need to be determined for each type of sample.

    3. Add 50 ul Buffer DS (and optional: 20 ul Proteinase K) to the lysate. Mix well by shaking. Proteinase K (20mg/ml) can increase the sensitive.

    4. Incubate at 65°C for 30-60 minutes. Mix sample several times during incubation by shaking.

    5. Centrifuge at 3,000-5,000 x g (5,000 x g is better, if available) for 10 min. Compact pellets will form at bottom of tubes but some particles may float. Be careful to avoid those particles during the transfer in next step.

    6. Carefully transfer 200 ul supernatant to 0.5 ml Plate, making sure not to disturb the pellet or transfer any debris.

    7. Add 20 ul Magsi Particles and 150 ul absolute thanol to each well. Mix well by pipetting up and down 50 times. Incubate at room temperature for 3 min.

    Note: Absolute ethanol and MagSi Particles can be premix. MagSi Particles will bead together in its container after several minutes It must be fully suspended by shaking or vortexing before use.

    8. Place the plate on a magnetic separation device to magnetize the MagSi particles. Remove and discard the cleared supernatant.

    9. Remove the plate containing the MagSi particles from the magnetic separation device. Add 400ul SPM Wash Buffer to each well.

    10. Resuspend MagSi particles pellet by by pipetting up and down 30 times. Place the tube on a magnetic separation device to magnetize the MagSi particles. Remove and discard the cleared supernatant.

    11. Remove the plate containing the MagSi particles from the magnetic separation device. Add 400ul SPM Wash Buffer to each well.

    12. Resuspend Magsi particles pellet by by pipetting up and down 30 times. Place the tube on a magnetic separation device to magnetize the Mag-Si particles. Remove and discard the cleared supernatant.

    13. Leave the plate to air dry on the magnetic separation device for 10min. Remove any residue liquid from tube by pipetting.

    14. Remove the tube from magnetic separation device. Add 30-50ul Elution Buffer or water to elute DNA from the magnetic particles.

    15. Resuspend MagSi particles by by pipetting up and down 30 times. Incubate 5 min at room temperature (best at 55-65°C). Mix well by pipetting up and down 30 times

    16. Place the tube onto a magnetic separation device to magnetize the MagSi particles. Transfer the cleared supernatant containing purified DNA to a new plate.

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