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Hybridization involves providing the conditions where probes can specifically anneal, or hybridize, with complementary nucleic acid sequences on the membrane.

After blotting, the membrane may be incubated with pre-hybridization solution containing blocking agents that minimize nonspecific binding of the probe to the membrane. The membrane is then incubated with the probe in hybridization buffer where the probe hybridizes to its complimentary nucleic acid target. These steps are usually performed in rotating tubes or bottles in a hybridization oven/shaker at a controlled temperature, often overnight. 

There are a number of factors that affect hybridization stringency. Temperature and salt concentration can be used to control the stringency, both during the hybridization and the subsequent washes that are used to remove unbound probe from the membrane. The hybridization temperature used for long DNA probes is usually 65°C. 

However for short probes, calculation of the probe fusion temperature (also known as Tm) is used to estimate the hybridization temperature. In some cases, it may be beneficial to utilize low stringency conditions, for example to detect low homology sequences (e.g., pseudogenes). After detection, the probe can be stripped from the membrane, so that it can be analyzed using other probes.

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