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    Mag-Binds Plant DNA Kit Magnetic 96 Protocol

    互联网

    883

    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

     

    Tissue Disruption

    Manual disruption:

    To prepare samples, collect plant sample in a 30 ml mortar and freeze by dipping in liquid nitrogen using tweezers or tongs to fill the tube. Grind the tissue using a clean pestle. Transfer the sample powder and liquid nitrogen into 1.2 ml 96 plate and allow the liquid nitrogen to evaporate. Immediately proceed with the DNA isolation protocol.

    Mechanical tissue disruption:

    Place sample into a stainless steel grinding jar with appropriate steel beads. Freeze samples in the stainless steel grinding jar using liquid nitrogen for 1 minute. Immediately attach the grinding jar onto the clamps of the Tissuelyser. Grind tissue at 30Hz for 1-2 minutes.

    1. Collect ground plant tissue (start with 50mg for fresh sample and 10mg for dried samples) in a 1.2 ml 96 well plate. Immediately add 600ul Buffer SLX Minus and 4ul RNase A. Shaking vigorously to disperse all clumps.

    Note: Buffer SLX Minus and RNase A can be combined in appropriate proportions to make a master mix before starting the procedure. RNase A activity is lost after longterm storage in Buffer SLX Minus.

    2. Incubate at 70°C for 10 minutes. Mix sample once during incubation shaking.

    3. 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.

    4. Carefully transfer 400 ul supernatant to 2ml Deep Well Plate, making sure not to disturb the pellet or transfer any debris.

    5. Add 20 ul MagSi Particles and 280 ul absolute thanol to each well. Shaking to mix for 2 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. (IMPORTANT)

    6. Place the plate on a magnetic separation device to magnetize the MagSi particles for 2-5 minutes . Remove and discard the cleared supernatant.

    7. Remove the plate containing the MagSi particles from the magnetic separation device. Add 500ul Buffer PHB to each well.

    8. Resuspend MagSi particles pellet by shaking for 2min. Place the tube on a magnetic separation device to magnetize the MagSi particles for 2-5 minutes. Remove and discard the cleared supernatant.

    9. Remove the plate containing the MagSi particles from the magnetic separation device. Add 500ul Buffer PHB to each well.

    10. Resuspend MagSi particles pellet by shaking for 2min. 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 500ul SPM Wash Buffer to each well.

    12. Resuspend MagSi particles pellet by shaking for 1 min. Place the tube on a magnetic separation device to magnetize the MagSi particles. Remove and discard the cleared supernatant.

    13. Remove the plate containing the MagSi particles from the magnetic separation device. Add 500ul SPM Wash Buffer to each well.

    14. Resuspend MagSi particles pellet by shaking for 1 min. Place the tube on a magnetic separation device to magnetize the MagSi particles. Remove and discard the cleared supernatant.

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

    16. Remove the tube from magnetic separation device. Add 50-100ul Elution Buffer or water to elute DNA from the magnetic particles.

    17. Resuspend MagSi particles by shaking for 1 min. Incubate 5 min at room temperature (best at 55-65°C). Shaking for 3 minutes.

    18. 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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