Bio-Rad Zeta-Probe Membranes - Manual (Page 7)

7. Disconnect the vacuum, disassemble the apparatus, and rinse
the membrane briefly in 2x SSC. UV-crosslink the DNA to the
membrane or vacuum dry the blotted Zeta-Probe membrane at
80°C for 30 min. The membranes can be stored dry between
two pieces of filter paper in plastic bags at 23–25°C.
2.6 RNA Dot/Slot Blotting
Both native and denatured RNA are retained quantitatively by
Zeta-Probe membrane. However, to insure optimal hybridization, RNA
samples must be totally denatured before fixing onto the Zeta-Probe
membrane.
Glyoxal RNA Denaturation and Fixation
1. Add RNA sample to the following final concentrations:
50% dimethyl sulfoxide (DMSO)
10 mM sodium phosphate, pH 7
1 M glyoxal
2. Incubate sample for 1 hr at 50°C. Then cool the RNA sample
on ice.
3. Wet a sheet of Zeta-Probe membrane by immersing it in
distilled water.
4. Assemble the microfiltration apparatus with the prewetted
Zeta-Probe membrane. Make sure that all the screws or clamps
have been tightened under vacuum to prevent cross well
contamination.
5. Place a 0.5 ml RNA sample into each appropriate well without
vacuum.
6. Apply vacuum until the wells are just dry, then release vacuum.
7. Rinse all wells with 0.5 ml TE, and apply vacuum until the wells
are completely dry.
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the membrane with filter paper. Fix nucleic acids onto the Zeta-Probe
membrane by baking it at 80°C for 30 min. The membranes can be
stored dry between two pieces of filter paper in plastic bags at
23–25°C.
2.5 DNA Dot Blotting
When Zeta-Probe membrane is used, it is not necessary to extract
DNA from tissue samples for dot blot analysis. Regardless of whether
the sample is purified DNA (covalently closed circular DNA, double-
stranded DNA, single-stranded DNA), whole blood, tissue, or cultured
cells, it can be heated in alkali, then filtered directly onto the
Zeta-Probe membrane.
1. Heat the sample in a total volume of 0.5 ml with a final concen-
tration equal to 0.4 M NaOH, 10 mM EDTA at 100°C for
10 min.
16 The sample may be purified or crude DNA ( ≤5 µg),
whole soft tissue, e.g., liver ( ≤0.5 mg), whole blood (≤10 µl),
cultured cells ( ≤10 5 cells).
2. Wet a sheet of Zeta-Probe membrane by immersing it in distilled
water.
3. Assemble the microfiltration apparatus with the prewetted
Zeta-Probe membrane. Make sure that all screws and clamps
have been tightened under vacuum to prevent contamination
between wells. Rinse wells with 0.5 ml TE or H
2
O. Apply vacuum
until wells are empty but not dry.
4. Apply a 0.5 ml DNA sample into each appropriate well without
vacuum.
5. Start vacuum until the wells are just dry.
6. Rinse all wells by placing 0.5 ml of 0.4 M NaOH in each, then
apply vacuum until all wells are quite dry.
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