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Kingfisher Flex 96 Plant DNA High Pure Protocol

互联网2013-11-13

1476

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 at least 3,000-5,000 x g

2. Rotor adapter for 96-well microplate

3. (Optional)Incubator equilibrated to 65°C

4. Equipment for disrupting Plant tissue (MM300 Mixer Mill or Geno/Grinder 2000 and Tungsten carbide beads) or Liquid Nitrogen

5. 8-or 12-channel pipette

6. Reagent reservoir for pipette

8. 1.5 or 2.0 mL microcentrifuge tubes, sealed deep-well plate or capped microtube rack for sample disruption

9. Kingfisher 96 or Kingfisher Flex 96 with Deep Well Magnet

10. 4 Deep Well Kingfisher 96 Plates

11. 1 Kingfisher 96 Plate

12. 1 Tip Sleeve for Kingfisher 96 Deep Well Magnet

13. Sealing Film

实验步骤

1. To prepare dried samples, place 10-50 mg of Plant into a deep well plate or 1.2 mL microtube rack in the presence of SLX Minus and grinding bead. Add 400ul Buffer SLX Minus and 2 uL of RNase A.

Note: SLX Minus Buffer 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. 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 the tube rack for 10-30 minutes at 70°C. Mix sample twice during incubation by inverting tube or vortexing plate very briefly.

4. Centrifuge at 3,000-12,000 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.

5. Carefully transfer 200ul supernatant to a 96-well Kingfisher Deep Well Plate, making sure not to disturb the pellet or transfer any debris.

6. Add 20ul/well of MagSi^® Particles Solution and 500ul Lysis Buffer to the sample.

7. Set up KingFisher Flex instrument by press “tart Key”on the Mag Bind Plant DNA Protocol and load plates according to prompts from Kingfisher Unit.

8. After the DNA isolation, seal Elution Plate which contains purified DNA with sealing Film (not provided) and store purified DNA at -20°C.

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