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Southern Blot, Have You Ever Heard About This Amazing Technique

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Before going into detail about what is a southern blot, First, let me give you a quick overview of actually what is blotting or blot.

Blotting is a technique that is being used in pathology laboratories for transferring RNA or DNA strand on a carrier. Most often this technique is done after the process of gel electrophoresis. It includes the transferring of DNA or RNA strands from the gel onto the blotting membrane. There are 4 types of blotting and southern blotting is one of em.

Types of blotting:

  1. Northern Blot.
  2. Eastern Blot.
  3. Western Blot.
  4. Southern Blot.

Let’s dive into detail about the southern blot.

What is Southern Blot ?

Southern blot is titled following Edward M. Southern. The technique is utilized for the investigation of DNA strands. Southern blot is a discovery method utilized to get the objective DNA strand present in the DNA compound sample in the domain of molecular biology.

The method begins from gel electrophoresis of DNA strands that are bonded in a blotting membrane accompanied by DNA transfer step from the gel is carried upon membrane that is being used in blotting.

Principle Of Southern Blot:

An enzyme called restriction endonuclease is employed to split the DNA to minute particles. These particles are later separated doing electrophoresis. The particles obtained are then classified on their size basis. Therefore, DNA particles are carried to the blotting paper where it is allowed to incubate with probes. Probes that are being employed in Southern blot can be selective extremely. They can selectively bond with the determination of 1 in a million and the properties to bond to the familiar targeted fragment.

Material Required For Southern Blot:

  • TAE or TBE buffer.
  • Agarose
  • Ethidium bromide, SYBR green,
  • DNA loading buffer
  • DNA markers
  • Hybridization mixture
  • Paraffin oil.
  • Nitrocellulose membrane
  • RNAse
  • Restriction enzyme
  • Radiolabelled RNA

Steps 

Step #1 

DNA is carried by using a proper RE. Restriction enzyme cleaves this DNA at a particular section making strands.

The quantity of DNA strand captured by RE is PCR amplified.`

Step #2

The DNA strand of interest is then isolated by the process gel electrophoresis.

Step #3

The electrophoresis gel is then dipped in an alkaline or acidic solution to denature the double-helical DNA strand.

After this DNA strand gets separated…

Step #4

The isolated DNA strand is later then carried to +vely charged nylon membrane by blotting.

Step #5

Later the DNA strand gets hybridized on the surface of the membrane, it is warmed on autoclave to fix on the nylon blotting membrane.

The nylon membrane is then employed with casein or Bovine serum albumin (BSA) which drenches all the membrane binding sites.

Step #6

The membrane hybridized DNA is then treated with a labelled probe.

The labeled probe includes the corresponding orders to the gene of concern

The probe bind with corresponding DNA on the membrane surface since all other non-specific binding site on the membrane has been prevented by casein or BSA.

Step #7

The DNA hybridized membrane labeled with the probe can be envisioned beneath the autoradiogram which gives the band pattern.

This is a basic overview of Southern Blot technique

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