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AuPreP™ DNA easy Plant Maxi kit

Cat. # PLX68-163LTD
Isolation of genomic DNA from 1 g plant material.

Kit Contents:

PX1 Buffer 90 ml
PX2 Buffer 30 ml
PX3 Buffer 110 ml
RNase A 85 mg
WS Buffer 50 ml
Plant Genomic DNA Maxi Column 20
Shearing tube 20
15ml Collection tube 40
Protocol 1
 

Notes:

  • All centrifugation should be done at room temperature with a swing-bucket centrifuge.
  • Preheat a water bath to 65°C.
  • Preheat TE or ddH2O to 65°C for DNA elution.
  • PX1 Buffer and PX3 Buffer may form a precipitate, warm at 65°C to redissolve.
  • Add 850µl of ddH2O to the RNase A powder tube, vortex to dissolve and store at 4°C.

Protocol:

  • Grind 1 g (or less) plant sample under liquid nitrogen to a fine powder and transfer to a new tube.
    Do not allow the sample to thaw, and continue immediately to step 2.
  • Add 4 ml of PX1 Buffer and 40 µl of RNase A solution (100 mg/ml) to the tissue powder and vortex vigorously, then incubate the mixture at 65°C for 10 minutes.
    Do not mix PX1 Buffer and RNase A prior to use. Invert 2-3 times during 65°C incubation.
  • Add 1.3 ml of PX2 Buffer to the lysate, vortex, and incubate on ice for 5 minutes.
  • Apply lysate to the Shearing tube sitting in a Collection tube and centrifuge at full speed (about 3000 rpm or 2500 x g) for 2 minutes. Transfer flow-through sample from the Collection tube to a new tube (not provided).
    Avoid pipetting any debris or pellet in the collection tube.
  • Add 0.5 volume of PX3 Buffer and 1 volume of 96-100% ethanol to the clear lysate and mix by pipetting.
    For example: If 4.5 ml clear lysate collected, add 2.25 ml PX3 Buffer and 4.5 ml ethanol.
  • Apply 6.5 ml of the ethanol added sample (including any precipitate) from step 5 to a Plant Genomic DNA Maxi Column sitting in a Collection tube, close the cap, centrifuge at full speed for 3 minutes, and discard the filtrate.
    If the solution remains above the membrane, centrifuge again.
  • Repeat step 6 for rest of the sample.
  • Wash the column twice with 5 ml of WS Buffer by centrifuging at full speed for 3 minutes and discard the filtrate.
    Add 200 ml of ethanol (96-100%) to the WS Buffer bottle when first open the bottle.
  • Centrifuge at full speed for 5 minutes to remove traces of WS Buffer.
  • Transfer the column to a new 15 ml tube (not provided), add 2 ml of 65°C TE or ddH2O, and centrifuge at full speed for 5 minutes to elute DNA.
  • Store DNA at -20°C.
 
 

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