Mag-Bind Plant RNA Protocol using 96-well Plate
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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 with swinging-bucket rotor for 96 well plates
3. 2 ml deep-well plate (Cat#EZ9602-02 )
4. 500 μl round well processing plate (Cat#EZ9604-02 )
5. Equipment for disrupting plant tissue (MM300 Mixer Mill or Geno/Grinder 2000)
6. Mortar and pestle (for manual tissue disruption)
7. Magnetic Stand for 96-well plate (Cat# MSD-01)
实验步骤
1. Collect ground plant sample (start with 15-20 mg) in a deep well process plate.
Note: For Kingfisher 96 instrument, the starting material can be doubled to 30-40 mg. Double all the reagent volume in this protocol.
2. Immediately add 300 uL Buffer MRPL and 10 uL Proteinase K solution (25mg/ml). Seal the plate with Sealing film and mix the sample thoroughly by vortexing at maximum speed for 30-60 seconds.
3. Add 100 uL Buffer SP2 and seal the plate with Sealing film vortex to mix throughly.
Note: It is critical to mix the sample throughly by vortexing vigorously to give optimized yield.
4. Incubate 10 minutes at -20°C. This step helps to remove the proteins, polysacchrides and other inhibitors.
5. Centrifuge at 4,000 x g for 20 min to pellet cell debris. Compact pellets will form in the plate but some particles may float. Be careful to avoid those particles while transferring the supernatant in next step.
6. Carefully transfer 200 uL of cleared supernatant to a 500 μl processing plate. Making sure not to disturb the pellet or transfer any debris.
7. Add 10uL Mag-Bind Particles Solution R follow by 210 uL of isopropanol. Mix by Pipetting up and down 10 times.
8. Incubate at room temperature for 5 minutes. Mix the sample 3 times during the incubation by pipetting.
9. Place the plate on magnetic stand (Cat# MSD-01) suitable for 96-well microplate to magnetize the Mag-Bind particles.
10. Wait until the magnetic beads form a pellet in the side of the wells (if using MSD-01) or until the solution is clear(if using another magnet). Aspirate and discard the cleared supernatant. Do not disturb the magnetic beads.
11. Add 300 uL of MRW Buffer Resuspend Mag-Bind™ particles pellet by pipetting up and down 10-20 times. Incubate 3 minutes at room temperature.
12. Wait until the magnetic beads form a pellet in the side of the wells (if using MSD-01) or until the solution is clear(if using another magnet). Place the plate onto a magnetic separation device to magnetize the Mag-Bind™particles. Aspirate and discard the cleared supernatant.
13. Remove the plate containing the Mag-Bind® particles from the magnetic stand. Add 300 uL of RWB Wash Buffer (diluted with ethanol) into the plate.
14. Resuspend Mag-Bind® particles pellet by pipettng up and down 10-20 times.
15. Place the plate onto a magnetic separation device to magnetize the Mag-Bind® particles. Wait until the magnetic beads form a pellet in the side of the wells (if using MSD-01) or until the solution is clear(if using another magnet) Remove and discard the cleared supernatant.
16. Remove plate from magnet and add 48uL DNase I Digestion Buffer and 1 uL DNase I. Resuspend the magnetic beads by pipetting or vortexing.
Note: If total nucleic acid (both DNA and RNA) are desired, skip the DNase I digestion step and proceed the wash step starting from step 20.
17. Incubate at room temperature (22-25°C) for 10 minutes.
18. Add 275 μl MRW Buffer to the tube and mix throughly by pipetting up and down for 20 seconds. Incubate 5 minutes at room temperature.
19. Place the plate on magnet stand to collect the magnetic beads. Wait until the magnetic beads form a pellet in the side of the wells (if using MSD-01) or until the solution is clear(if using another magnet) Carefully remove and discard the cleared supernatant.
20. Remove the plate from magnet and add 300 μl RWB Buffer. Resuspend the magnetic beads by vortexing.
21. Place the plate on magnet stand to collect the magnetic beads. Wait until the magnetic beads form a pellet in the side of the wells (if using MSD-01) or until the solution is clear(if using another magnet) Carefully remove and discard the cleared supernatant
22. Leave the plate on the magnet to air dry the magnetic beads for 5-10 minutes. Remove any residue liquid from tube by pipetting.
23. Remove the plate from magnetic separation device. Add 50-100 ul DEPC-treated water and resuspend the beads by pipetting.
24. Incubate 5-10 minutes at room temperature. Place the plate onto magnet to magnetize the Mag-Bind particles. Wait until ® the magnetic beads form a pellet in the side of the wells (if using MSD-01) or until the solution is clear(if using another magnet)
25. Transfer the cleared supernatant containing purified RNA to a new 96-well microplate.
