Colony Hybridization

This techniquesis 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 hybridizaion experiment the procedure for colony hybridization is briefly described below.
(!) The bacterial cells subjected to transformation areplated onto a suitable agar plate this is the master plate.
(2) The colonies of master plate are replica plated onto a nitrocellulose filter membrane placed on agar medium. For replica a block of wood or cork, of suitable diameter for the master plate, is covered with velvet cloth. This block sterlized and the lowered into the master plate till the velvet touches all the colonies the block is withdrawn and gently lowered onto the nitrocellulose filter so that bacterial cells sticking onto the velvet are transferred onto the filter.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 the on 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 denature theDNA released fromthese 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 back at tofix 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.