GENE CLONING

Tutorial

1. Why both of the DNA of interest and vector DNA should be restricted by same restriction enzyme?

2. List out the cloning process.

3. How to test the efficiency of recombination?

4. Why the transformants can survive during antibiotic screening?

 

Answer: 

1. To generate similar restriction site. Same restriction site enable the ligation process can be carried out more easily.

2.a. Choose the suitable DNA of interest and the cloning vector.

   b. Restrict both of the DNA samples with same restriction enzyme.

   c. Mix and incubate the restricted DNA in the presence of ligase.

   d. Insert the recombinant vector into the host cell.

   e. Screening can be carried out to study the efficiency of transformation and recombination.

3. By blue-white screening. Non-recombinant DNA containing functional Lac Z gene that will produce functional

    β- galactosidase. Functional β- galactosidase can cleave the X-gal and produce blue pigment. While, recombinant      

    DNA cannot produce functional Lac Z gene. Hence, no X-gal will be cleaved and appear as white colonies.

4.Transformants carry the plasmid vector containing antibiotic- resistant gene. Antibiotic-resistant gene protects the

   transformants from being destroyed by antibiotics.