Clone libraries You have a mixed culture of bacteria and the

Clone libraries You have a mixed culture of bacteria, and there are several versions of a given biodegradation gene that you would like to sequence. All of the genes contain sequences targets for the restriction enzymes Eco RI and Hindi, which bracket your gene of interest. Give a step-by-step description of how you would use a clone library approach, using the plasmid pUC19, to sequence the gene of interest. 5 or 6 steps should be sufficient, and you can stop with blue/white screening as your last step.

Solution

pUC19 is a cloning vector .

1.In order to clone a piece of DNA into the vector , we need to first cleave the piece of DNA with the restriction enzyme, such that the gene of interest would be obtained.

2. The vector is cleaved with the same Restriction enzyme so as to obtain sticky ends onto which the required GOI can insert itself.

3. This vector is then inserted into the bacterial cells.

4.The bacterial cells have been made ready to take up the planks by treatment with Calcium phosphate, and then ready to be transformed.

5. These calls are then plated onto a plate with IPTG and XGal supplemented with ampicillin in order to select for the recombinant colonies.

6.The colonies that have taken up the recombinant plasmid will be white in colour and the ones with plasmid but insertion in region except MCS will produce blue coloured colonies .