Bio-Rad Zeta-Probe Membranes - Manual (Page 11)

2x SSPE
1% (w/v) SDS
0.5% (w/v) BLOTTO
10% (w/v) dextran sulfate
0.5 mg/ml nonhomologous carrier DNA
4.4 Oligonucleotide Protocol 6
Prehybridization
1. Seal the blotted Zeta-Probe membrane inside a heat-sealable
plastic bag. Prepare the following solution for prehybridization:
5x SSC
20 mM Na
2
HPO
4
, pH 7.2
7% SDS
1x Denhardt’s
100 µg/ml denatured herring sperm DNA
The carrier DNA must be denatured before adding it to the
hybridization solution by heating at 100°C for 5 min, followed by
rapid cooling on ice.
2. Cut one corner of the plastic bag and pipet prehybridization
solution in, then reseal the bag.
3. Incubate at 50°C for 0.5–24 hr.
Hybridization
1. Immediately before use, fragment and denature the probe and
carrier DNA as follows. Dissolve the radiolabeled probe in
0.1 ml of 0.2 M NaOH, add carrier DNA, mix, and centrifuge
briefly to consolidate the solution. Pierce a fine hole in the tube
cap and place the tube in a heating block at 100°C for 5 min,
followed by rapid cooling on ice.
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Note: For single-copy detection or high stringency, conduct the
last wash at 65°C.
2. After washing, the blotted membranes are ready for autoradiogra-
phy. If no further cycles of hybridization are to be done on the
membrane, the membrane can be dried. When reprobing, do not
allow the membrane to dry between hybridizations. Expose moist
membranes between plastic wrap or enclosed in a sealable plastic
bag. Do not allow a wet membrane to come in contact with the
film, because wet Zeta-Probe membrane will stick to the film.
4.3 Alternative Protocol
In this section two hybridization protocols using hybridization
accelerators are presented. When extreme hybridization sensitivity is
needed, these accelerators will help to increase the target signal by
acting as volume excluders. Hybridization accelerators will also
decrease the hybridization time needed. In some applications,
hybridization accelerators can reduce the hybridization time from
overnight to 4 hr. It is suggested that you first work with the standard
hybridization protocol (Section 4.1) and determine if your experiments
require a hybridization accelerator before using the following protocols.
1. Polyethylene glycol (PEG) 15 — follow the instructions for standard
hybridization (Section 4.1) or formamide hybridization (Section 4.2)
except add 10% (w/v) PEG 8,000 MW into the hybridization solu-
tion in step 1.
Conduct post-hybridization washes as described in Section 4.1 or
4.2, without PEG.
2. Dextran sulfate — follow the instructions for formamide hybridiza-
tion (Section 4.2) except increase the hybridization temperature to
65°C and substitute the following prehybridization and hybridiza-
tion solutions in step 1:
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