Nucleic acid hybridization
After blotting, the probe is incubated with the blot in hybridization buffer where it hybridizes to its complimentary target on the membrane. 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 oligos, calculation of the probe fusion temperature (also known as "Tm") is used to estimate the hybridization temperature.
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