Question

For identification of desired D by Southern blotting, select the correct order
A. Blotting
B. Probe binding
C. Autoradiogram
D. Electrophoresis
E. Gel treatment

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

Hint:

Southern Blotting Protocol Steps:
1. Electrophoresis (D) $\to$ 2. Gel Treatment/Deturation (E) $\to$ 3. Membrane Blotting (A) $\to$ 4. Probe Hybridization (B) $\to$ 5. Autoradiography Detection (C).

Answer 1

The Correct Option is B

Step 1: Understanding the Question:
The question asks for the proper sequential order of experimental steps carried out during Southern blotting for target D detection.

Step 2: Key Formula or Approach:
Southern blotting involves digesting target D with restriction enzymes, separating fragments by gel electrophoresis, deturing D, transferring single strands to a membrane, hybridizing with labeled probes, and detecting bands via imaging.

Step 3: Detailed Explation:

•

Step 1: Electrophoresis (D): Cleaved D fragments are separated according to molecular size on an agarose gel.

•

Step 2: Gel Treatment (E): The gel is treated with alkaline solution ($\text{OH}$) to deture double-stranded D into single-stranded D.

•

Step 3: Blotting (A): Single-stranded D is transferred from the gel onto a solid support membrane (nitrocellulose or nylon) via capillary or vacuum blotting.

•

Step 4: Probe Binding (B): The membrane is incubated with a labeled single-stranded probe to allow complementary hybridization.

•

Step 5: Autoradiogram (C): Unbound probes are washed away, and hybridized probe-target bands are visualized using autoradiography or chemiluminescent imaging.

Step 4: Fil Answer:
The correct experimental sequence is D, E, A, B, C.