Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus - Manual (Page 15)

Section 7
Hybridization Protocols for Nucleic Acids
7.1 Probe Recommendations
The specific activity, concentration, size range, and purity of the probe all have an important
effect on signal-to-noise ratio during hybridization. For hybridization on Zeta-Probe membrane,
the following is recommended:
Probe specific activity: 108 cpm/µg probe
Probe concentration in
the hybridization mixture:
106 counts/ml (10–50 ng/ml)
Probe length: 200–1,000 b p
Optimal probe specific activity and concentration can vary according to available hybridization
sites and exposure time. Alternative hybridization protocols are necessary when probe lengths
vary outside this recommended range (Bio-Rad Laboratories 1987).
Probe cleanup is essential to minimize background. Unincorporated nucleotides present after
probe preparation contribute to hybridization background. The most effective cleanup method is
by column chromatography. This can be done quickly and easily with the Bio-Spin
®
chromatography columns (Bio-Spin 6 columns, or Bio-Spin 30 columns, catalog number 732-
6004).
After cleanup, denature double-stranded probes by heating to 95–100°C for 5 minutes. Then cool
rapidly on ice. Use the probe as soon as possible after preparation.
There are several hybridization protocols given in this section. All protocols are for using DNA
probes to hybridize to either DNA or RNA. The 7% SDS hybridization protocol requires minimal
prehybridization treatment and has a high signal strength and low background. Further
references and techniques for hybridizing to the Zeta-Probe membrane may be found in the
Zeta-Probe membrane instruction manual.
The final volume of hybridization solution is important in reducing background. For
prehybridization and hybridization, use 150 µl solution/cm 2 of membrane. For washes, use at
least 350 µl/cm 2 of membrane.
7.2 Hybridization Protocols for DNA or RNA Bound to Nitrocellulose or
Zeta-Probe
® Membrane
Prehybridization
1. Place the blotted membrane inside a heat-sealable plastic bag. Seal three sides, leaving the
top side open.
2. Pipet in the correct prehybridization solution for your application:
For DNA or RNA Bound to For DNA Bound to For RNA Bound to
Zeta-Probe Membrane Nitrocellulose Nitrocellulose
1 mM EDTA 6x SSC 50% formamide
7% SDS 0.5% SDS 5x SSC
0.5 M NaHPO
4
, pH 7.2 5x Denhardt’s solution 1x Denhardt’s solution
100 µg/ml denatured 50 mM NaHPO
4
, pH 6.5
salmon sperm DNA 250 µg/ml denatured
1 mM EDTA salmon sperm DNA
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