Bio-Rad Zeta-Probe Membranes - Manual (Page 9)

The T
m
is decreased approximately 1.5°C for every 1% decrease in
homology. 10, 11
The T
m
is decreased as the fragment length of the probe decreases;
the appropriate correction factor is approximately –500 / (# bp in
probe fragment)°C.
10, 11
The rate of hybridization increases as the salt concentration
increases.
10
The rate of hybridization decreases as the formamide concentration
increases.
10, 13
The hybridization temperature (TH) appropriate to synthetic oligomeric
DNA probes in 1 M Na+ can be approximated by the following:
T
H
+ 2 x (no. of A-T bp) + 4 x (no. of G-C bp) –5.14
4.1 Standard Protocol
Prehybridization
1. Seal the blotted Zeta-Probe membrane inside a heat-sealable
plastic bag.
2. Cut one corner of the plastic bag and pipet prehybridization
solution in:
0.5 M Na
2
HPO
4
, pH 7.2
7% (w/v) SDS
3. Incubate briefly at 65°C for 5 min. The goal is to evenly and
completely coat the membrane with this solution.
Hybridization
1. Cut one corner of the plastic bag, remove the prehybridization
solution, and replace it with the same buffer.
13
hybridization to be performed at a lower temperature. The protocol in
Section 4.4 is recommended for oligonucleotide probes. The
Alternative Protocol (Section 4.3) should be used only when extreme
sensitivity is needed.
The final volume of hybridization solution is important in reducing
background. For prehybridization, use 150 µl solution/cm
2 Zeta-Probe
membrane. For washes, use at least 350 µl solution/cm 2 Zeta-Probe
membrane.
One of the most significant advantages offered by Zeta-Probe
membrane over conventional membranes is that target nucleic acids
of all sizes can be fixed irreversibly. The stringency of hybridization can
therefore be optimized for detection of specific target sequences.
There is no need to use high ionic strength and low temperature to
minimize the loss of nucleic acids from the membrane during
hybridization or washing procedures.
Hybridizations should be conducted at 20–25°C below the melting
temperature (T
m
) of the probe duplex to insure optimal rates of specif-
ic hybridization while minimizing interaction with partially homologous
sequences.
10 The stringency of post-hybridization washes is less criti-
cal, but a good rule of thumb is to conduct the most stringent wash at
10–15°C below T
m
. 11 The protocols described below are suitable for
probes having a (G+C) content representative of the mammalian
genome, i.e., 0.42. If desired, conditions can be varied in accordance
with the following empirical formula:
T
m
(DNA/DNA) = 81.5 + 16.6 x log [Na] – 0.65 x (% formamide) + 41 x
(G + C).11
T
m
(RNA/RNA) = 79.8 + 18.5 x log [Na+] – 0.35 x (% formamide) +
58.4 x (G + C) + 11.8 x (G + C)2. 12
T
m
(DNA/RNA) = approx. mean of T
m
(DNA/DNA) and T
m
(RNA/RNA)
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