Bio-Rad Aurum™ Total RNA Mini Kit User Manual
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13
Animal and Plant Tissue
Follow steps D1–D5, then continue with step 1 of "All Starting Sample Types"
on page 13.
D1. Cut the tissue into small pieces (<5 mm long) and grind it into a fine
powder with a mortar and pestle containing liquid nitrogen. Make sure
that the tissue does not thaw by periodically adding liquid nitrogen to
the mortar.
D2. Transfer up to 20 mg hard animal tissue, up to 40 mg soft animal tissue,
or up to 60 mg plant tissue into an RNase-free 2.0 ml capped
microcentrifuge tube (provided).
Note: Plant tissue requires the Aurum lysis solution to be supplemented
with 2% (w/v) polyvinylpyrrolidone-40 (PVP). For each column processed,
add 14 µl PVP to every 700 µl of lysis solution before proceeding to step 3.
D3. Add 700 µl of lysis solution to the tube. Resuspend the sample by
pipetting up and down. Use a rotor-stator homogenizer for 30–60 sec or
other equivalent method to disrupt the sample.
D4. Centrifuge the lysate for 3 min and transfer the supernatant into a new
2.0 ml capped microcentrifuge tube (provided).
D5. Add 700 µl of 70% ethanol for plant tissue or 60% ethanol for animal
tissue to the supernatant. Mix thoroughly by pipetting up and down or by
using a rotor-stator homogenizer. Make sure that no bilayer is visible.
All Starting Sample Types:
1.
Attach an Aurum total RNA binding column to a luer fitting of the column
adaptor plate on the Aurum vacuum manifold or to a compatible vacuum
manifold. Refer to Figure 2 for setup. The vacuum source should be
turned off and the vacuum regulator should be completely open.
2. Decant or pipet the homogenized lysate into the RNA binding column.
Turn the vacuum on and adjust to –17 to –23 inHg by closing the
vacuum regulator. Continue to apply vacuum until all the lysate has
passed through the column. Open the vacuum regulator until the gauge
indicates 0 inHg.
3.
The low stringency wash solution is provided as a 5x concentrate. Add
4 volumes (80 ml) of 95–100% ethanol to the low stringency
wash solution concentrate before initial use.
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