Question

Select the correct order of steps involved in Cloning:
A. Selection
B. Restriction digestion
C. Ligation
D. Transformation
E. Gene of Interest (Insert) and vector

• A. E, B, D, A, C
• B. E, D, B, C, A
• C. E, B, C, D, A
• D. E, C, D, A, B

Hint:

Cloning Core Steps:
1. D/Vector (E) $\to$ 2. Cut/Digest (B) $\to$ 3. Paste/Ligate (C) $\to$ 4. Transform (D) $\to$ 5. Select (A).

Answer 1

The Correct Option is C

Step 1: Understanding the Question:
The question asks for the standard chronological sequence of steps involved in gene cloning using recombint D technology.

Step 2: Key Formula or Approach:
Recombint D cloning involves isolating target D and vector, cutting both with restriction enzymes, joining them covalently, introducing the recombint construct into host cells, and screening transformed colonies: Isolate target D/Vector (E) $\rightarrow{}$ Restriction Cleavage (B) \to D Ligation (C) $\rightarrow{}$ Host Transformation (D) $\rightarrow{}$ Colony Selection (A)

Step 3: Detailed Explation:

• 1. Target D and Vector Preparation (E): Obtaining the gene of interest (insert D) and selecting an appropriate cloning vector (such as pBR322 or pUC plasmid).

• 2. Restriction Digestion (B): Cleaving both the target D insert and vector plasmid using complementary restriction endonucleases to generate matching sticky or blunt ends.

• 3. Ligation (C): Incubating digested insert and vector D together with T4 D Ligase to form phosphodiester bonds, generating recombint D molecules.

• 4. Transformation (D): Introducing recombint plasmids into competent host bacterial cells (e.g., E. coli) via heat-shock or electroporation.

• 5. Selection (A): Plating host bacteria onto selective media (containing antibiotics like ampicillin or X-gal/IPTG for blue-white screening) to identify recombint transformants.

Step 4: Fil Answer:
The correct step sequence in molecular cloning is E, B, C, D, A.