Blotting is used in molecular biology to transfer nucleic acids and proteins from gel to a membrane for identification and analysis. Developed in the 1970s, it combines electrophoresis and immunological methods. There are three main types: Southern (DNA), Northern (RNA), and Western (proteins), each allowing detection and measurement of specific molecules.
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Biotechnology, Molecular Microbiology, Techniques in Microbiology Lab
Western blotting technique or protein immunoblot is used to identify specific proteins separated according to their sizes in an electrophoresis experiment. Protein immunoblot unlike other blotting techniques (e.g. southern and northern blotting) utilizes specific antibodies to identify the protein of interest. It is the transfer of proteins from gel to a matrix or nitrocellulose membrane.
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Biotechnology, Molecular Microbiology
Northern blotting technique is used to detect specific sequences of ribonucleic acid (RNA). The protocol for performing northern blotting technique is similar to that of southern blotting technique. Northern blotting technique was first described by James Alwine and colleagues in 1977 as a molecular biology technique for identifying specific nucleotide sequence of a piece of
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Biotechnology, Molecular Microbiology
Southern blotting, developed by Sir Edward M. Southern in 1975, is a molecular technique used to detect specific DNA sequences. It involves transferring DNA from a gel to a nitrocellulose membrane, followed by hybridization with radiolabeled probes. This method is pivotal in DNA analysis, forensic science, and paternity testing.
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Biotechnology, Molecular Microbiology
Melting curve analysis and detection systems Melting curve analysis can only be performed with realtime PCR detection technologies in which the fluorophore remains associated with the amplicon. Amplifications that have used SYBR® Green I or SYBR® GreenER™ dye can be subjected to melting curve analysis. Dual-labeled probe detection systems such as TaqMan® probes are not
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Biotechnology, Molecular Microbiology, Techniques in Microbiology Lab
TaqMan® probe signal production Whether an MGB or non-MGB probe is chosen, both follow the same pattern for signal production. In the early PCR cycles, only the low, quenched reporter signal is detected. This early signal, automatically subtracted to zero in the real-time PCR software, is termed “baseline”. If the sample contains a target, eventually
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Biotechnology, Molecular Microbiology, Techniques in Microbiology Lab
Real-time PCR fluorescence detection systems Real-time fluorescent PCR chemistries Many real-time fluorescent PCR chemistries exist, but the most widely used are 5” nuclease assays such as TaqMan®Assays and SYBR® Green dye–based assays (Figure 1). The 5” nuclease assay is named for the 5” nuclease activityassociated with Taq DNA polymerase (Figure 2). The 5” nuclease domain
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Biotechnology, Molecular Microbiology, Techniques in Microbiology Lab
BaselineThe baseline of the real-time PCR reaction refers to the signal level during the initial cycles of PCR, usually cycles 3 to 15, in which there is little change in fluorescent signal. The low-level signal of the baseline can be equated to the background or the “noise” of the reaction (Figure 1). The baseline in
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Biotechnology, Molecular Microbiology, Techniques in Microbiology Lab
Good primer design is one of the most important parameters in real-time PCR. This is why many researchers choose to purchase TaqMan® Assay products—primers and probes for real-time PCR designed using a proven algorithm and trusted by scientists around the world. If you choose to design your own real-time PCR primers, keep in mind that
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Biotechnology, Molecular Microbiology, Techniques in Microbiology Lab
DNA polymerasePCR performance is often related to the thermostable DNA polymerase, so enzyme selection is critical to success. One of the main factors affecting PCR specificity is the fact that Taq DNA polymerase has residual activity at low temperatures. Primers can anneal nonspecifically to DNA during reaction setup, allowing the polymerase to synthesize nonspecific product.
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Molecular Microbiology, Techniques in Microbiology Lab