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Mag-Binds Plant DNA Kit Magnetic Fast Protocol

互联网2013-11-13

796

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 plant tissue (start with 50mg for fresh sample and 10mg for dried samples) in a 1.2 ml 96 well plate with appropriate steel beads. Add 600ul 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. Incubate at 70°C for 10 minutes. Mix sample once during incubation by shaking.

4. Centrifuge at 3,000-5,000 x g (5,000 x g is better, if available) for 15 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.

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

6. 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.

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

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

9. Resuspend Magsi particles pellet by shaking for 1 min. Place the tube on a magnetic separation device to magnetize the MagSi particles. Removeand discard the cleared supernatant.

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

11. 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.

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

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

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

15. 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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