Question

If the vector was cut using HindIII, what would colonies growing on a medium containing tetracycline DEFINITELY contain?

(b) Suggest which of the three restriction enzymes would be suitable for insertion of the gene of interest. Give TWO advantages of using the enzyme chosen by you.

(c) State ONE disadvantage of using the other two restriction enzymes not chosen in (b).

Answer 1

(a) The uncut vector without the gene of interest

(b) EcoRI would be ideal to use:

It has a single restriction site, meaning that only two products will be obtained - either with the gene of interest or without it and will not give multiple products.

The restriction site is within the chloramphenicol resistance gene, which on insertional inactivation will help select recombinant vectors.

(c) Using ScaI and/or HindIII would lead to multiple end products/fragments being formed which would make selection of the recombinant vector complicated.