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Prehybridization prepares the membrane for probe hybridization by blocking non-specific nucleic acid-binding sites on the membrane. This ultimately serves to lower background. Many different prehybridization solutions have been described in the literature. However, the prehybridization solutions described here combine efficient blocking with ease of use. As with any probe, optimal hybridization conditions for DIG-labeled probes must be determined experimentally. We strongly recommend that the time be taken to optimize each DIG-labeled probe (see the mock hybridization). The time taken for optimization will result in cleaner results and, ultimately, time savings, especially if a probe will be reused many times.
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Originally nitrocellulose membranes were used but their fragility prompted to search for alternative types of support matrix, resulting in the introduction of nylon membranes in the early 1980s. The main advantages of nylon membranes is their greater tensile strength and that DNA can be bound covalently by UV cross-linking. Nylon membranes can therefore be reprobed up to about 12 times without becoming broken or losing their bound DNA. In contrast nitrocellulose membranes are fragile (allow only 3 times reprobing) and do not bind DNA covalently: baking to immobilize the DNA leas to a relatively weak hydrophobic attachment through exclusion of water. The disadvantage of nylon membranes is the amount of background signal seen after hybridization. On the other site nylon membranes are able to bind about five times more DNA per cm2 than nitrocellulose. Nylon retains DNA fragments down to 50 nucleotides in length, but nitrocellulose is inefficient with molecules
With nitrocellulose the transfer buffer must provide a high ionic strength to promote binding of the DNA to the membrane. Several formulations exists but 20 x SSC is recommended because it is easy to make up and can be stored for several months at room temperature. Lower SSC concentrations (e.g. 10x) should not be used with nitrocellulose as the lower ionic strength may result in loss of smaller DNA fragments during transfer. Alkaline transfer is not suitable for nitrocellulose as they do not retain DNA at pH 9.0 and fall apart after long exposure to alkali.
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Please review this section of general hybridization considerations before proceeding with the DIG-system. Several points are critical for successful use of the DIG-system, especially when performing chemiluminescent detection.
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: If chemiluminescent detection is performed, a too high probe concentration will often lead to background. Therefore, the probe concentration should not be increased above the recommended concentrations.
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The DIG System is an effective system for the labeling and detection of DNA, RNA, and oligonucleotides. The protocols for labeling with digoxigenin (Figure 1) and subsequent detection are based on well established, widely used methods. DNA, RNA, and oligonucleotide probes are labeled according to the methods (usually enzymatic) used for preparing radioactive probes. Hybridization of digoxigenin-labeled probes (e.g., to target DNA or RNA on a Southern or Northern blot) is also carried out according to standard protocols, except that a special blocking reagent is used to eliminate background. The signal on the nucleic acid blot is detected according to the methods developed for western blots. The incorporation and spacing of digoxigenin in DNA, RNA, and oligonucleotides can be varied by using different labeling protocols.
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Figure 7: Mock hybridization and effect probe concentration. Naked pieces of membrane were incubated with the indicated amounts of DIG-labeled DNA probe and detected with chemilumininescence.
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The hybridization incubation is carried out in a high-salt solution that promotes base-pairing between probe and target sequences. Hybridization is normally carried out below the Tm for the probe/target and the specificity of the experiment is the function of post-hybridization washes. The critical parameters are the ionic strength of the final wash solution and the temperature at which this wash is done.