By Dilip Kumar Arora, Surajit Das, Mesapogu Sukumar
This Springer Protocols guide is a pragmatic consultant to the appliance of key molecular biology concepts in microbiological examine. the point of interest is on experimental protocols, that are offered in an easy-to-follow method, as step by step tactics for direct use within the laboratory. Notes on how one can effectively practice the strategies are incorporated, in addition to suggestions relating to fabrics and providers. as well as the sensible protocols, vital heritage details and consultant result of experiments utilizing the defined equipment are provided. Researchers in all parts using microbial structures, comparable to in molecular biology, genetics, pathology, and agricultural examine will locate this paintings of serious value.
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Additional info for Analyzing Microbes: Manual of Molecular Biology Techniques
SYBER Green binds double stranded DNA and upon excitation emits light. An increase in DNA product during PCR leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified (Fig. 4). The advantages of SYBER Green are that it is inexpensive, easy to use, and sensitive. However, dsDNA dyes such as SYBR Green binds to all dsDNA PCR products, including nonspecific PCR products (such as “primer dimers”). This can potentially interfere with/or prevent accurate quantification of the intended target sequence.
Denaturation at 95 C for 15 s. 2. Annealing/extension at 60 C for 1 min (repeated 40 times). 4. 1. 2. Probe Selection Criteria Whenever possible, primers and probes should be selected in a region with a G/C content of 30–80 %. Regions with G/C content in excess may not denature well during thermal cycling, leading to a less efficient reaction. In addition, G/C-rich sequences are susceptible to nonspecific interactions that may reduce reaction efficiency and produce nonspecific signal in SYBR Green assays.
The more times the denaturing and annealing takes place, the more opportunities there are for the TaqMan probe to bind and, in turn, the more emitted light is detected, and its geometric increase corresponding to exponential increase of the product is used to determine the threshold cycle (Ct) in each reaction. Fluorescent reporter probes are more accurate and reliable of the method. It uses a sequence-specific RNA or DNA-based probe to quantify only the DNA containing the probe sequence; 26 Praveen and Koundal therefore, use of the reporter probe significantly increases specificity, and allows quantification even in the presence of some nonspecific DNA amplification.
Analyzing Microbes: Manual of Molecular Biology Techniques by Dilip Kumar Arora, Surajit Das, Mesapogu Sukumar