Identification of c-DNA containing plasmid:

 

 

Identification of c-DNA containing plasmid:

For selection of c-DNA containing plasmid, a lot of techniques are used.One of the important technique is colony hybridization technique.It is developed by Grunstein and Hogness in 1975.The method is as follows:

 

1. The transformed bacterial culture is diluted and poured onto an agarized medium in a petridish. The culture plate is incubated till bacterial colonies appear.

 

2. A nitrocellulose filter is placed over the colonies on the agarized medium for sometime. Some cells from each of the colonies get adsorbed on the filter and form a replica of the master plate. The position is marked.

 

3. The master plate is retained as such. The nitrocellulose filter is taken from the petridish and placed in alkali solution (0.5N NaOH). The alkali solution lyses the bacterial cells and denatures the DNA as well.

 4. The filter is kept dipped in a solution containing proteinase K, an enzyme that removes protein from the filter. As a result, DNA alone gets bound to the filter.

 5. The DNA is fixed firmly on the filter by drying the filter at 80°C inbetween filter papers. This step is called baking. Consequently, the filter has DNA print of the bacterial colonies.

 6. The filter is placed in a hybridization solution containing DNA probe or RNA probe labelled with P ³². The probe combines with the exact target DNA in the DNA print and forms a DNA-DNA hybrid or RN A-DNA hybrid.

 7. The filter is then washed with standard saline citrate solution to remove unbound probes.

 8. An X-ray film is placed over the filter for about 4 hours to get an autoradiogram. The probe makes dark spots on the X-ray film.

9. The autoradio gram is matched with the master plate. The black spots in the autoradiogram locate the exact position of recombinant colonies in the master plate. The recombinant cells contain the particular desired gene.

l0. The recombinant colonies are picked up and cultured for future use.