Arrange the following steps involved in transformation of bacteria in a sequence from initiation to end.

(A) Incubation of rDNA with bacterial cell on ice

(B) Treatment with divalent cations

(C) Heat shock treatment

(D) Selection on antibiotic-containing agar plate

(E) Placed them again on ice

Choose the correct answer from the options given below:

(B), (A), (C), (E), (D)

The correct answer is Option (2) - (B), (A), (C), (E), (D)

Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. In order to force bacteria to take up the plasmid, the bacterial cells must first be made ‘competent’ to take up DNA.

This is done by treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.

Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42⁰C (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.

The transformed bacterial cells are plated on an agar medium containing an antibiotic. Only the cells that have successfully taken up and expressed the antibiotic resistance gene (often included in the rDNA) will survive and grow, allowing for the selection of transformed colonies.