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
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
What is Nycodenz Nycodenz is a non-ionic, triiodinated radiopaque substance used primarily in molecular biology and biochemistry for density gradient centrifugation. Its chemical formula is C_19H_26I_3N_3O_9. Nycodenz creates gradients that are used to separate cells, viruses, organelles, and other biological particles based on their density. Nycodenz is a brand name for a non-ionic, iso-osmotic density
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Environmental & Soil Microbiology, Microbe Lab, Techniques in Microbiology Lab
Luria-Bertani (LB) broth is the most widely used medium for the growth of bacteria. It is a standard medium for propagation of Escherichia coli. It can also be used when culturing cells for plasmid preparation. LB is a nutrient-rich microbial broth that contains peptides, amino acids, water-soluble vitamins, and carbohydrates. It is also suitable for non-selective
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Culture media, 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
Real-time PCR is a variation of the standard PCR technique that is commonly used to quantify DNA or RNA in a sample. Using sequence-specific primers, the number of copies of a particular DNA or RNA sequence can be determined. By measuring the amount of amplified product at each stage during the PCR cycle, quantification is
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Molecular Microbiology, Techniques in Microbiology Lab