Q.51 Arrange the steps involved in cloning of gene into plasmid:

1. Selection of confirmation of recombinant clones
2. Ligation of the gene of interest and plasmid
3. Amplification of the gene of interest using template DNA
4. Transformation of bacterium using the recombinant plasmid
5. Restriction endonuclease treatment of the suitable plasmid and gene of interest

Choose the correct answer from the options given below:

1. C, E, B, D, A
2. E, D, C, A, B
3. B, A, E, C, D
4. C, B, A, D, E

   Gene cloning into plasmid follows a standard molecular biology protocol with precise sequential steps. Option 1 correctly orders them for recombinant DNA technology exams like GATE Life Sciences.

   Question Breakdown

   Steps (labeled as per options):

   • C: Amplification of the gene of interest using template DNA (PCR to generate insert).

   • E: Restriction endonuclease treatment of the suitable plasmid and gene of interest (digestion for compatible ends).

   • B: Ligation of the gene of interest and plasmid (joining insert to vector).

   • D: Transformation of bacterium using the recombinant plasmid (introducing into host like E. coli).

   • A: Selection of confirmation of recombinant clones (screening via antibiotics, blue-white, PCR).

   Correct Sequence

   C → E → B → D → A

   1. Amplify gene (C) via PCR.

   2. Digest both gene and plasmid (E) with restriction enzymes.

   3. Ligate (B) to form recombinant plasmid.

   4. Transform bacteria (D).

   5. Select/confirm recombinants (A).

   Answer: Option 1 (C, E, B, D, A)

   Option Analysis

   • Option 1 (C, E, B, D, A): Correct. Logical flow: amplify → digest → ligate → transform → select.

   • Option 2 (E, D, C, A, B): Wrong. Digestion (E) before amplification (C) impossible; no gene to digest. Ligation (B) last illogical.

   • Option 3 (B, A, E, C, D): Wrong. Ligation (B) first without fragments; selection (A) before transformation (D) premature.

   • Option 4 (C, B, A, D, E): Wrong. Ligation (B) before digestion (E); selection (A) before transformation (D).​

   Gene cloning into plasmid steps sequence is essential for molecular biology exams like GATE Life Sciences. This guide details the correct order—amplification, restriction endonuclease treatment, ligation, transformation, selection—for Q.51 mastery.

   Step 1: Amplification (C)

   PCR amplifies the gene of interest from template DNA, producing thousands of copies with flanking restriction sites. Essential first step for sufficient insert DNA.​

   Step 2: Restriction Digestion (E)

   Treat gene PCR product and plasmid with same restriction endonucleases (e.g., EcoRI) to create sticky ends. Allows directional cloning; often followed by gel purification.

   Step 3: Ligation (B)

   T4 DNA ligase joins compatible ends of insert and linearized plasmid, forming circular recombinant DNA. Ratio ~3:1 insert:vector optimizes success.​

   Step 4: Transformation (D)

   Heat-shock or electroporate competent E. coli with recombinant plasmid. Cells take up DNA; grown on selective media (e.g., ampicillin).​

   Step 5: Selection/Confirmation (A)

   Screen colonies: antibiotic resistance selects transformants; blue-white screening (X-gal) or PCR confirms inserts. Miniprep verifies by digestion.

   Step	Action	Purpose
   C	PCR amplification	Generate insert ​
   E	Restriction digestion	Compatible ends ​
   B	Ligation	Recombinant formation ​
   D	Bacterial transformation	Host propagation
   A	Clone selection	Verify recombinants ​