By Dilip Kumar Arora, Surajit Das, Mesapogu Sukumar
This Springer Protocols guide is a realistic advisor to the applying of key molecular biology recommendations in microbiological examine. the point of interest is on experimental protocols, that are offered in an easy-to-follow means, as step by step techniques for direct use within the laboratory. Notes on find out how to effectively observe the systems are integrated, in addition to techniques concerning fabrics and providers. as well as the sensible protocols, vital historical past info and consultant result of experiments utilizing the defined tools are provided. Researchers in all parts employing microbial structures, resembling in molecular biology, genetics, pathology, and agricultural study will locate this paintings of significant value.
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Extra info for Analyzing Microbes: Manual of Molecular Biology Techniques
Scorpions In Scorpion probes, sequence-specific priming and PCR product detection are achieved using a single oligonucleotide. The Scorpion probe maintains a stem–loop configuration in the unhybridized state. The fluorophore is attached to the 50 end and is quenched by a moiety coupled to the 30 end. The 30 portion of the stem also contains sequence that is complementary to the extension product of the primer. This sequence is linked to the 28 Praveen and Koundal Fig. 7 Molecular beacon; a hairpin fluorescent probe.
Molecular beacons are designed so that their probe sequence is just long enough for a perfectly complementary probe–target hybrid to be more stable than the stem hybrid. The length of the probe sequence (10–40 nt) is chosen in such a way that the probe target hybrid is stable in the conditions of the assay. The stem sequence (5–7 nt) is chosen to ensure that the two arms hybridize to each other but not to the loop sequence (Fig. 7). The computer program is used to predict melting temperature of the stem and also to predict whether the intended stemand-loop conformation will occur or not.
For instance, concentrations of reaction components such as MgCl2, primer, and dNTPs as well as the quality and concentration of the target DNA have to be experimentally tested for amplification efficiency. Once these parameters are adapted for the organism under study, PCR reactions can be set up. In RAPD, only one primer is used (unlike the traditional 48 Reineke and Uma Devi two for standard PCR reactions). RAPD PCR reactions are performed at very low annealing temperature and a high number of PCR cycles—usually 45 cycles are applied.