6. Centrifuge for 20 min at maximum speed in a microcentrifuge. Transfer the supernatant to a fresh 1.5 ml or 2 ml microcentrifuge tube (not supplied).
A minimum g-force of 10,000 x g is required.
Transfer of small debris remaining in the supernatant after centrifugation at full speed will not affect the procedure.
7. Add 350 µl 95% ethanol. Mix by vortexing, and centrifuge briefly (1–2 s; £1000 x g) to remove drops from the inside of the tube lid.
Note: The length of the centrifugation must not exceed 1–2 s, as this may result in pelletting of nucleic acids and reduced yields of total RNA.
use 95% ethanol; 100% ethanol often contains benzene (bad for arrays).
8. Apply 700 µl sample to the PAXgene column sitting in a 2 ml processing tube, and centrifuge for 1 min at ³8000 x g (³10,000 rpm). Place the PAXgene column in a new 2 ml processing tube, and discard the old processing tube containing flow-through.
9. Apply the remaining sample to the PAXgene column, and centrifuge for 1 min at ³8000 x g (³10,000 rpm). Place the PAXgene column in a new 2 ml processing tube, and discard the old processing tube containing flow-through.
We do not perform the optional DNAse step.
10. Apply 700 µl Buffer BR3 to the PAXgene column, and centrifuge for 1 min at ³8000 x g (³10,000 rpm). Place the PAXgene column in a new 2 ml processing tube, and discard the old processing tube containing flow-through.
11.
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