User Manual

4. Assemble the Bio-Dot apparatus according to the instructions in Section 3.1.
Remember to apply the vacuum and then retighten the screws that hold the apparatus
together.
5. Samples and wash solutions may be applied with a standard pipet or a Costar
Octapette pipet. Apply the denatured RNA, and pull the sample through by passive
filtration or by applying a gentle vacuum.
Note: a method for applying gentle vacuum to the apparatus is to adjust the flow rate
valve to setting 3. Use a finger to cover the valve port exposed to air. The amount of
vacuum reaching the manifold will be regulated by the pressure of your finger on the
valves.
6. Rinse all wells to wash through any sample on the side of the wells. Rinse with 500 µl
TE. Apply vacuum (flow valve setting 1, Figure 3) until the sample wells are dry.
7. Disassemble the Bio-Dot apparatus. Remove the blotted membrane.
8. Remove glyoxal adducts by pouring 20 mM Tris-HCl, pH 8.0, 1 mM EDTA heated to
95°C onto the membrane and agitating at room temperature until the solution cools.
Place the membrane in a vacuum oven at 80°C for 1 hour for the Zeta-Probe membrane,
2 hours for nitrocellulose.
9. Nitrocellulose membrane must be baked under vacuum for 2 hours at 80°C before
hybridization. If hybridization is not to be undertaken within 2 days, then bake the
Zeta-Probe membrane under vacuum for 30 minutes at 80°C. The Zeta-probe membrane
and nitrocellulose membranes can be stored dry between two pieces of filter paper in
plastic bags at 23–25°C.
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