By Dilip Kumar Arora, Surajit Das, Mesapogu Sukumar
This Springer Protocols handbook is a pragmatic consultant to the applying of key molecular biology options in microbiological learn. the point of interest is on experimental protocols, that are awarded in an easy-to-follow approach, as step by step tactics for direct use within the laboratory. Notes on how you can effectively practice the methods are incorporated, in addition to thoughts relating to fabrics and providers. as well as the sensible protocols, very important historical past info and consultant result of experiments utilizing the defined tools are provided. Researchers in all parts using microbial structures, resembling in molecular biology, genetics, pathology, and agricultural study will locate this paintings of serious value.
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This Springer Protocols handbook is a pragmatic advisor to the appliance of key molecular biology suggestions in microbiological study. the focal point is on experimental protocols, that are offered in an easy-to-follow manner, as step by step strategies for direct use within the laboratory. Notes on tips on how to effectively follow the methods are incorporated, in addition to concepts concerning fabrics and providers.
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Extra resources for Analyzing Microbes: Manual of Molecular Biology Techniques
Dynabeads from Dynal), washed according to the manufacturer’s instructions 8. TA cloning vector kit 9. Competent Escherichia coli cells 10. 5. SSCP (Single Strand Conformation Polymorphism) 1. 6. ARDRA (Amplified Ribosomal DNA Restriction Analysis) 1. One forward and one reverse primer for amplification of the specific target sequence 2. 7. T-RFLP (Terminal Restriction Fragment Length Polymorphism) 1. Fluorescently labelled primers (see Note 5). If both primers used are labelled, a different dye is used for each.
1 Introduction Restriction endonucleases are a class of enzymes that cut DNA molecules. Each enzyme recognizes a unique sequence of nucleotides in the DNA strand, usually about 4–6 base pairs long. The sequences are palindromic in that the complimentary DNA strand has the same sequence only in the reverse direction, so both strands of DNA are cut at the same location . Restriction enzymes are found in many different strains of bacteria where their biological role is to participate in cell defense.
The process is based on the detection of the fluorescence produced by a reporter molecule which increases, as the reaction proceeds. This occurs due to the accumulation of the PCR product with each cycle of amplification. The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle. Here we discuss the detailed explanation of various fluorescent molecules and strategies to determine the viral load.