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Colony Hybridization - This technique is used to identify those bacterial colonies in a plate, which contain a specific DNA sequence. These bacterial colonies are obtained from bacterial cells into which this sequence was introduced through genetic engineering, and the given sequence is represented by the probe used in the hybridization experiment. The procedure for colony hybridization is briefly described below.

1. The bacterial cells subjected to transformation are plated onto a suitable agar plate; this is the master plate.

2. The colonies of master plate are replicaplated onto a nitrocellulose filter membrane placed on agar medium. For replica plating, a block of wood or cork, of suitable diameter for the master plate, is covered with velvet cloth.

This block is sterilized and then lowered into the master plate till the velvet touches all the colonies; the block is withdrawn and gently lowered onto the nitrocellulose filter so that the bacterial cells sticking on to the velvet are transferred onto the filter. The master plate is retained intact for later use. A reference point is marked both on the master plate and on the replica plate to facilitate later comparisons.

3. After the colonies appear, the filter is removed from the agar plate and treated with alkali to lyse the bacterial cells. This also denatures the DNA released from these cells.

4. The filter is treated with proteinase K to digest and remove the proteins; the denatured DNA remains bound to the filter.

5. The filter is now baked at 80° C to fix the DNA; this yields the DNA-print of bacterial colonies in the same relative positions as those of the colonies themselves in the master plate.

6. The filter is now hybridized with the radioactive probe; the probe represents the sequence of DNA segment used for transformation.. The unhybridized probe is removed by repeated washing.

7. The colonies whose DNA hybridizes with the probe are detected by autoradiography; only these colonies show up in the autoradiograph.

The positions of colonies showing up in the autoradiograph are compared with the masterplate to identify the colonies that contain the DNA segment in question. The colonies are then picked up for further studies. A variation of this procedure can be applied to phage plaques as well.

 
 

 
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