By Dilip Kumar Arora, Surajit Das, Mesapogu Sukumar
This Springer Protocols guide is a realistic consultant to the applying of key molecular biology innovations in microbiological study. the point of interest is on experimental protocols, that are provided in an easy-to-follow means, as step by step techniques for direct use within the laboratory. Notes on how one can effectively follow the techniques are incorporated, in addition to ideas concerning fabrics and providers. as well as the sensible protocols, vital history details and consultant result of experiments utilizing the defined equipment are offered. Researchers in all components utilizing microbial structures, reminiscent of in molecular biology, genetics, pathology, and agricultural learn will locate this paintings of serious value.
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Extra info for Analyzing Microbes: Manual of Molecular Biology Techniques
Size of ampilcon should be <500 bp. Small amplicons are favored because they promote high-efficiency assays. In addition, high-efficiency assays enable relative quantification to be performed using the comparative method or threshold cycle (Ct). This method increases sample throughput by eliminating the need for standard curves when looking at expression levels of a target relative to a reference control. 5. General Recommendations for Real-Time RT-PCR The optimal concentrations of the real-time PCR reagents are as follows: 1.
Two specifically designed, sequence-specific oligonucleotide probes, labeled with different dyes, are used. The sequences of the probes are selected so that they can hybridize to the target sequences on the amplified DNA fragment in a head-to-tail orientation, thus bringing the two dyes into close proximity. The donor dye (fluorescein) is excited by the blue light source and emits green fluorescent light at a slightly longer wavelength. At close proximity, the energy emitted excites the acceptor dye attached to the second hybridization probe, which then emits fluorescent light at a different wavelength.
LightCycler® 480 Sealing Foil. 4. 2Â Light CyclerR 480 SYBR Green 1 master mix/TaqMan probe. 5. Gene-specific primers. 2. QRT-PCR Reaction Requirements (Using TaqMan Probe) Total RNA (as CMV is a RNA virus) from the infected plant leaf samples 1. 0 ml 2. 0 ml 3. 0 ml 4. 0 ml 5. 5 ml 6. 5 ml 7. 0 ml 8. 5 ml 9. 5 ml 10. 3. Cycling Parameters 1. Reverse transcription (using M-MuLV) at 48 C for 30 min. 2. Taq activation 95 C for 10 min. PCR profile: 1. Denaturation at 95 C for 15 s. 2. Annealing/extension at 60 C for 1 min (repeated 40 times).
Analyzing Microbes: Manual of Molecular Biology Techniques by Dilip Kumar Arora, Surajit Das, Mesapogu Sukumar