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2.1.5.1 Colony hybridization
It is the preferred choice for screening the colonies to identify and isolate the colony which contains the desired gene.
- In this technique the cells are first plated on selective plates.
- A replica of the colonies is made on nitrocellulose filter disc which is placed on the surface of a second plate.
- The colonies are allowed to grow on the master plate and the nitrocellulose disc.
- The disc is then removed and placed in alkali to lyse the bacteria in situ and to denature their DNA.
- The single-stranded DNA binds to the nitrocellulose filter in the position originally occupied by the bacterial colony.
- The filter is than baked at 800C, following which it is incubated with a solution containing the radiolabelled c DNA probe under conditions which favour nucleic acid hybridization.
- The unhybridized material is removed by extensive washing, thus allowing the identification of colonies containing sequence s complementary to the probe by autoradiography .
- Colonies which give a positive autoradiograph signal can then be picked from the master plate and cultured in order to provide sufficient cells carrying the desired gene.
Last modified: Friday, 22 June 2012, 5:22 AM