Colony Hybridization

This technique is used to identify those bacterial colonies in a plate which contain a specific DNA sequence.

• The recombinant bacterial cells are plated onto a suitable agar plate; this is the “Master plate”.
• The sterilized vector cloth containing block cork are lowered into the master plate till the velvet touches all the colonies; the block is withdrawn and gently lowered onto the nitrocellulose filter, the bacterial cells sticking on to the velvet are transferred onto the filter.
• A reference point is marked both on the master plate and the on replica plate to facilitate later comparisons.
• The nitrocellulose 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.
• The filter is treated with “Proteinase.K” to digest and remove the proteins; the denatures DNA remains bound to the filter.
• The filter is now backed at 80⁰C to fix the DNA; in this the DNA-print in bacterial colonies in the same relative positions in the master plate.

 

• 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.
• The colonies, whose DNA hybridizes with the probe are detected by “Auto radiography”, only these colonies show up in the autoradiography.
• The positions of colonies showing up in the autoradiography are compared with the master plate to identify these colonies; these colonies contain the DNA segment. The colonies are then picked up for further studies.

See the Video Tutorial

Colony Hybridization