What is the purpose of vector only control for the ligation

What is the purpose of vector only control for the ligation reaction? Will vector itself ligate and form a circular DNA? Will it form colonies on Kanamycin plate? Why or why not?

Solution

Please find the answers below:

What is the purpose of vector only control for the ligation reaction?

Answer: A vector is used to carry the gene of interest along with accessory markers which can be transported to a target cell and transfer the gene of interest in the target DNA. This vector contains a selectable marker and reporter gene which help in screening the positive ligates and ensuring that the gene of interest has indeed been transferred to the target DNA. However, since every transformation/transfection reaction is not 100% successful, some of the cells remain un-transformed/un-transfected. Similarly, the vector is also a DNA which can itself exert any genetic manipulation effect on the target DNA, although it is not desirable that the vector induces any genetic manipulation in the target DNA. In order to ascertain this as an experimental proof, researchers carry a negative control experimental group in order to ascertain that the vector itself does not induce any manipulation. Thus, the purpose of using a vector only control for the ligation reaction as a negative control.

Will vector itself ligate and form a circular DNA?

Answer: The vector is a DNA sequence which may or may not have self-complimentary sticky ends depending upon the nature of restriction enzyme it has been digested with. If the restriction enzyme produces sticky ends such as that digested with EcoRI etc, the vector will indeed form a circular DNA. However, if a restriction enzyme produces blunt ends, it will not undergo self-ligation and not produce circular DNA.

Will it form colonies on Kanamycin plate? Why or why not?

Answer: The vector often contains an antibiotic resistance gene which is incorported in order to ascertain that the vector has been ligated and select the positive transformation colonies in the culture. However, for this anti-biotic gene to get expressed, the vector must be ligated in the genomic DNA. Thus, mere addition of the vector control will not lead to expression of this vector DNA and thus kanamycin resistance will not be provided. Thus, these colonies will not grow on kanamycin plate.