Question

describe southern blotting in detail i.Stepwise explain​

Answer 1

Answer:

Southern blotting is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.

Explanation:

Step 1: Restriction digest

• The DNA is fragmentized by using suitable restriction enzyme. RE cuts the DNA at specific site generating fragments.
• The number of fragments of DNA obtained by restriction digest is amplified by PCR

Step 2: Gel electrophoresis

The desired DNA fragments is separated by gel electrophoresis

Step 3: Denaturation

• The SDS gel after electrophoresis is then soaked in alkali (NaOH) or acid (HCl) to denature the double stranded DNA fragments.
• DNA strands get separated

Step 4: Blotting

The separated strands of DNA is then transferred to positively charged membrane nylon membrane (Nitrocellulose paper) by the process of blotting.

Step 5: Baking and blocking

• After the DNA of interest bound on the membrane, it is baked on autoclave to fix in the membrane.
• The membrane is then treated with casein or Bovine serum albumin (BSA) which saturates all the binding site of membrane

Step 6: Hybridization with labelled probes

• The DNA bound to membrane is then treated with labelled probe.
• The labelled probe contains the complementary sequences to the gene of interest.
• The probe bind with complementary DNA on the membrane since all other non-specific binding site on the membrane has been blocked by BSA or casein.

Step 7: Visualization by Autoradiogram

• The membrane bound DNA labelled with probe can be visualized under autoradiogram which give pattern of bands.

Answer 2

Answer:

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Explanation:

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