Southern Blot Test: Correct Stepwise Procedure for DNA Analysis
The steps involved in the Southern Blot test should be performed in the following order 1 – x-ray film 2 = electrophoresis 3 = digestion with restriction enzyme 4 = ethidium bromide 5 = radioactive probe
A. 32451
B. 34251
C. 32541
D. 24351
What Is the Correct Order of Steps in the Southern Blot Test?
The correct answer is:
✅ A. 3 → 2 → 4 → 5 → 1 (32451)
The Southern blot test is a molecular biology technique used to detect specific DNA sequences within a mixture of DNA fragments. The process involves multiple steps, each crucial for ensuring accurate detection of the target sequence.
Step-by-Step Process of Southern Blotting
-
DNA Digestion with Restriction Enzymes (Step 3)
- The first step involves cutting the DNA sample into smaller fragments using restriction enzymes. These enzymes recognize specific sequences and cleave the DNA at those sites, producing restriction fragments of varying sizes.
-
Electrophoresis (Step 2)
- The digested DNA fragments are then subjected to gel electrophoresis, where they migrate through an agarose gel under an electric field.
- Smaller fragments travel faster than larger ones, leading to their separation based on size.
-
Staining with Ethidium Bromide (Step 4)
- The gel is treated with ethidium bromide, a dye that intercalates between DNA bases and fluoresces under UV light.
- This step allows visualization of DNA fragments to confirm successful electrophoretic separation.
-
Hybridization with a Radioactive Probe (Step 5)
- The DNA fragments are transferred onto a nitrocellulose or nylon membrane through a capillary or vacuum transfer system.
- A radioactively labeled DNA probe, complementary to the target sequence, is then introduced. The probe binds to the specific DNA fragment containing the sequence of interest.
-
Detection Using X-ray Film (Step 1)
- To visualize the hybridized probe, the membrane is exposed to x-ray film (autoradiography).
- Dark bands appear on the film, indicating the presence of the target DNA sequence.
Why Are the Other Options Incorrect?
- B (34251) and C (32541) – These sequences incorrectly place ethidium bromide or hybridization steps before the correct order.
- D (24351) – Incorrect placement of digestion and staining disrupts the process.
Conclusion
The Southern blot test follows a strict order: DNA digestion, electrophoresis, staining, probe hybridization, and detection. This technique is fundamental in genetic research, forensic science, and medical diagnostics, helping identify gene mutations, hereditary disorders, and viral integrations in DNA.
68 Comments
Suman bhakar
March 8, 2025Done
Aakansha Sharma
August 20, 2025Option Ais right
Khushi Vaishnav
August 19, 2025DNA digestion with restrictive enzyme – gel electrophoresis – Staining with Et B -Hybridization by Radioactive Probe -detection through x-ray film .
Dipti Sharma
August 20, 2025Dna digestion, electrophoresis, staining, probe hybridization, and detection.
A. 3 → 2 → 4 → 5 → 1
Mahima Sharma
August 21, 20253,2,4,5,1
Khushi Mehra
August 20, 2025Correct order is 32451
Digestion by using restriction enzyme , Electrophoresis , EtBr, radioactive probe, and R- ray film
Dharmpal Swami
August 20, 2025Option A is write answer
Mansukh Kapoor
August 20, 2025The correct answer is option 1st
Bharti Yadav
August 20, 20251.DNA digestion with restriction enzymes
2. Gel electrophoresis
3. Staining
4.probe hybridization
5.detection
Option 1 is correct
Sakshi yadav
August 20, 2025Option 1
Surbhi Rajawat
August 20, 2025The steps if Southern blotting are
DNA digestion
Gel electrophoresis
Staining
Probe binding
Detection of DNA
Priya dhakad
August 20, 20251.Digestion with Restriction enzyme
2 . Electrophoresis
3. Eithidium bromide
4.radioactive probe
5. X-ray film
anjani sharma
August 20, 2025Option 1 would be correct
Neelam Sharma
August 20, 2025A
The steps if Southern blotting are
DNA digestion
Gel electrophoresis
Staining
Probe binding
Detection of DNA
Deepika Sheoran
August 20, 2025Option A is correct answer
Digestion with Restriction
Electrophoresis
Ethidium bromide
Radioactive probe
X Ray film
Neha
August 21, 2025Dna digestion
Electrophoresis
Staining
Probe
X-ray film
Sakshi
August 22, 2025A is correct option
Firstly digestion of DNA by restriction enzyme, then gel electrophoresis is done bands are not visible on the gel then idiom bromide strain is given to visualize the bands ethidium bromide strain is give to visualise the bands in gel ,then radioactive prob is given to the membrane, and then observe the bands under x-ray
Mohd juber Ali
August 21, 2025Option (a)
1.Digestion (RE)
2.gel electrophoresis
3.staining
4. Radioactive probe
5. X ray film
Aman Choudhary
August 21, 2025Option A is correct
Digestion by Restriction Endonuclease ➡️ Gel electrophoresis ➡️ EtBr ➡️ Radioactive probe ➡️ x-ray film
Manisha
August 21, 2025DNA digestion,
electrophoresis,
staining,
probe hybridization,
X ray film and detection
Aafreen Khan
August 21, 2025Steps of Southern Blotting are
1 =Digestion with restriction enzyme
2 = Gel electrophoresis
3 = ethidium bromide
4 = radioactive probe
5 = X-Ray film
Avni
August 21, 2025A. 3 → 2 → 4 → 5 → 1
Dna digestion, electrophoresis, staining, probe hybridization, and detection.
Bhawna Choudhary
August 21, 2025Firstly Digestion with restrictions enzyme then electrophoresis then ethidium bromide then radioactive probe then x_ray film
Santosh Saini
August 21, 2025Digestion with restriction enzymes – electrophoresis – ethidium bromide – radioactive – x-ray film
Nilofar Khan
August 22, 2025Correct answer is a
3.digetion with restriction enzyme
2.electroforesis
4ethidium bromide
Radioactive probe
1.xray film
Simran Saini
August 21, 20253,2,4,5,1
Digestion with restriction enzyme, gel electrophoresis, ethidium bromide, radioactive probe, x-ray film.
Mitali saini
August 22, 2025A. 3 → 2 → 4 → 5 → 1 (32451)
Priyanka choudhary
August 22, 2025Right answer is option A,
Dna digestion
Electrophoresis
Staining
Probe
X-ray film
Sakshi Kanwar
August 22, 2025A …. DNA is provided with restriction enzyme first then gel electrophoresis is performed , the medium is provided with ethidium bromide , probe is given for hybridization at last x ray filming is performed to observe the hybridized band
Soniya Shekhawat
August 22, 2025👍
karishma
August 22, 20251 – x-ray film 2 = electrophoresis 3 = digestion with restriction enzyme 4 = ethidium bromide 5 = radioactive probe- answer is 3-2-4-5-1
Parul Yadav
August 22, 2025Steps of Southern Blotting:
(1) Electrophoresis
(2) Staining with Ethedium Bromide
(3) Hybridization with radio labeled probe
(4) Detection using X- ray film
Arushi
August 22, 2025Restriction -gel-stain-probe-xray
Kirti agarwal
August 23, 2025Opt a
Priya khandal
August 23, 202532451 done sir
Akshita Jangid
August 23, 2025Option A is correct answer 3,2,4,5,1
Devika
August 24, 2025Digestion, electrophoresis,stain, radioactive probe,xray film
Option A
Yogita
August 24, 202532451
Sneha Kumawat
August 24, 2025Isolate -restriction enzyme – gel electrophoresis -ethedium bromide- radioactive probe – detection by x ray film
Urmi sisodiya
August 24, 2025DNA digest with the help of restriction enzyme, electrophoresis, provide ethidium bromide afterwards radioactive probe and last xray film
AKANKSHA RAJPUT
August 24, 20253 – 2 – 4 – 5 – 1
Meenakshi Choudhary
August 24, 20251.Digestion with Restriction enzyme
2 . Electrophoresis
3. Ethidium bromide
4.radioactive probe
5. X-ray film
Kajal
August 24, 2025The steps are as follows:-
1. Digestion
2. Electrophoresis
3.Ethidium bromide
4. Radioactive probe
5.x-ray film
Shivani
August 24, 20251.Digestion with Restriction enzyme
2 . Gel Electrophoresis
3. Staining by Ethidium bromide
4.radioactive probe
5. Detection on X-ray film
So option A. Is correct
Ankita Pareek
August 24, 2025Option A is correct
Bhavana kankhedia
August 24, 202532451
Anisha Beniwal
August 24, 2025Option A
Neeraj Sharma
August 24, 2025Option A is correct
Ayush Dubey
August 24, 2025Option A is correct
Himanshi
August 24, 20251.Digestion with Restriction enzyme2 . Electrophoresis3. Ethidium bromide4.radioactive probe5. X-ray film
Tanvi Panwar
August 24, 2025Correct sequence is – digestion with restriction enzymes, electrophoresis,ethidium bromide, radioactive probe, x-ray film.
Surendra Doodi
August 24, 20251.DNA digestion with restriction enzymes
2. Gel electrophoresis
3. Staining
4.Probe hybridization
5.Detection
Priyanka Verma
August 24, 2025option A is correct
32451
Sonam Saini
August 24, 2025Right sequence 32451
sakshi khatri
August 24, 202532451 is correct order
Falguni Prakash
August 24, 202532451 option a
Kajal
August 25, 2025Option A is right
Priyanka Meena
August 26, 20251. Restriction enzyme
2. Gel electrophoresis
3.erithum bromide
4. Radioactive probe
5. X rale film
Manisha choudhary
August 27, 2025Aa. Option is correct answer
3. Digestion with restriction endonuclease – 2. Electrophoresis – 4. Eithidium bromide – 5. Probe – 6. Xray
Kanica Sunwalka
August 28, 2025done
Komal Sharma
August 30, 2025Option first is correct order is 32451
Shriyanshi verma
August 30, 2025Dna digestion, electrophoresis, staining, probe hybridization, and detection.
A. 3 → 2 → 4 → 5 → 1
Asha Gurzzar
August 30, 2025A is correct,3-2-4-1 ,dna digestion, electrophoresis, staining with ethidiun bromide,probe hybridisation,and detection
Alec
August 30, 2025option A is correct – 3,2,4,5,1.
Pratibha Jain
August 30, 2025The correct answer is option
A. 3 → 2 → 4 → 5 → 1 (32451)
Anisha jakhar
August 31, 20253-2-4-5-1
Shobha Kanwar
August 31, 2025Dna digestion, electrophoresis, staining, probe hybridization, and detection.
A. 3 → 2 → 4 → 5 → 1
Anjali
September 1, 2025A is correct answer