Molecular Biology

Southern Blotting Process

Southern blotting is a hybridization technique for identification of particular size of DNA from the mixture of other similar molecules.
Steps in southern blotting process include:

DNA extraction.
Restriction Digestion or DNA Fragmentation.
Agarose Gel Electrophoresis
Denaturation and Depurination
Blotting
Baking and blocking
Hybridization
Washing
Detection

Southern Blotting

Southern blotting is a hybridization technique for identification of particular size of DNA from the mixture of other similar molecules. This technique is based on the principle of separation of DNA fragments by gel electrophoresis and identified by labeled probe hybridization.
It combines transfer of electrophoresis separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization, which is a key process.

Western Blotting

An immunoblotting technique which rely on the specificity of binding between a protein of interest and a probe (antibody raised against that particular protein) to allow detection of the protein of interest in a mixture of many other similar molecules is western blotting.
It is used for the detection of proteins and also called protein immunoblotting because an antibody is used to specifically detect its antigen.

Detectors in Real Time PCR

Real time PCR employs detector for the monitoring of the progress of reaction (amplification of DNA). Detectors used can be various fluorescence markers. Among them the most common and important ones includes Taqman Probe and SYBR Green. Taqman assay is a widely used RT-PCR assay because of its sensitivity and specificity. It is based on 5′–3′ exonuclease activity of Taq DNA polymerase. Nonspecific fluorescent dye SYBR works on principle that when SYBR dyes bind to double-stranded DNA, their fluorescence increases.

Real Time PCR (Principle)

Real Time PCR is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR).  The term “real time” denotes that it can monitor the progress of the amplification when the process is going on. It is used to monitor the progress of a PCR reaction in real time. At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified.