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Genes and genomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Touchdown PCR is a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3°C-5°C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3°C-5°C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.
Effect of nitrogen limitation on polyhydroxyalkanoates production efficiency, properties and microbial dynamics using a soil-derived mixed continuous culture
Published in International Journal of Biobased Plastics, 2019
Ioanna Ntaikou, Ioannis Koumelis, Maria Kamilari, Zacharoula Iatridi, Constantinos Tsitsilianis, Gerasimos Lyberatos
Total DNA extraction, using 106 cells on the average, was carried out using the Macherey-Nagel Tissue kit following the manufacturer’s protocol. The genetic diversity and similarity of the bacterial community was analyzed by amplification of the 16S rRNA marker (corresponding to positions 341–534 in E. coli), using eubacteria-specific primers, as described by Muyzer et al. [14]. PCRs were carried out in 50 μL volumes (1 unit KAPA Taq DNA Polymerase, 1X KAPA PCR buffer A, 0.2 mM dNTPs, 1 mM MgCl, 0.5 μL DNA template, filled to 50 μL with sterile H2O). The thermocycling program for the touchdown PCR was as follows: initial denaturation was performed at 93°C for 5 min and then at 93°C for 1 min, followed by touchdown primer annealing from 65°C to 53°C (the annealing temperature was decreased 0.5°C every cycle for 25 cycles, to touchdown at 53°C), followed by extension at 68°C for 1 min (for each of the 25 cycles), with a final extension step at 68°C for 10 min. The PCR results were analyzed by horizontal electrophoresis in 1% agarose gel stained with ethidium bromide (1 μg/ml), after which they were inspected under UV light and photographed. Furthermore, in order to detect subtle differences in the amplified fragments, a second electrophoresis was performed in a 3% gel for 4 h.
Effect of Benzophenone-3 on performance, structure and microbial metabolism in an EGSB system
Published in Environmental Technology, 2020
Laura Castrillón Cano, Yudy Andrea Londoño, Nancy J. Pino, Gustavo A. Peñuela
For Archaea domain, primer PARCH340F (5′-CCCTACGGGG(C/T)GCA(G/C)CAG-3′) with a GC-clamp (5′CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG-3′) and PARCH519R (5′-TTACCGCGGC(G/T)GCTG-3′) were used [31]. Touchdown PCR was performed with the following conditions: 92°C for 2 min; 11 cycles of denaturation/annealing/extension with 1 min at 92°C for denaturation, 1 min at the annealing temperature, and 1 min at 72°C for the extension step. The initial annealing temperature was set at 64°C and was decreased by one degree every cycle, to reach the touchdown temperature at 54°C. The PCR procedure was completed after 20 cycles at 54°C annealing temperature.