- 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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