Cell disruption process is carried out in presence of buffer which maintains the integrity of biomolecules within the cell so it is called Extraction Buffer. They are also called as recovery buffer as these are used during recovering the desired nucleic material, proteins etc. by disrupting the cells.
The composition of buffer used for extraction is an essential part for disrupting cell to release its components. Common components of recovery buffer includes antioxidants, EDTA, enzyme substrate and cofactors, enzyme inhibitors, sodium azide, Polyvinylpyrrolidone etc.
The process of transferring of the denatured fragments out of the gel and onto a membrane made from nylon (or sometimes nitrocellulose) where they become accessible for analyzing using a probe is blotting. Blotting is a method of transferring proteins, DNA, RNA onto a carrier (special membrane). This membrane may be nitrocellulose, PVDF or nylon membrane.
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:
Restriction Digestion or DNA Fragmentation.
Agarose Gel Electrophoresis
Denaturation and Depurination
Baking and blocking
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 involves following steps: Protein Extraction /Sample preparation, Electrophoresis, Blotting and Probing.
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.
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.
The working procedure of Real Time PCR can be divided in two steps: Amplification and Detection. Amplification involves multiplication of template DNA.
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.