User Manual

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: 10
8
cpm/µg probe
Probe concentration in
the hybridization mixture: 10
6
counts/ml (10–50 mg/ml)
Probe length: 200–1,000 bp
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, catalog number 732-6000, 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 application:
For DNA or RNA Bound to For DNA Bound to For RNA Bound to
Zeta-Probe Membrane Nitrocellulose Nitrocellulose
(Bio-Rad Laboratories 1987) (Maniatis et al. 1982) (Thomas 1980)
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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