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Molecular Analysis in Mechanobiology
Published in Jiro Nagatomi, Eno Essien Ebong, Mechanobiology Handbook, 2018
Because SYBR green detection is nonspecific, validation procedures are required to ensure the amplification of a single product with desired sequence. The simplest method of validation is melting curve analysis, which should be included at the end of every reaction [45]. To generate a melting curve after the last cycle of amplification, the instrument incrementally increases the temperature from the detection to denaturation temperature while monitoring sample fluorescence. Separation of the dsDNA product at its melting temperature leads to an abrupt decline in fluorescence. The product melting temperature is readily identified as a peak in a plot of the negative rate of change of sample fluorescence with respect to temperature. For a valid reaction, there should be one peak corresponding to one product. Melting curve analysis can also reveal the presence of primer-dimers. Due to their shorter length, primer-dimers appear as small peaks at relatively lower temperatures than amplicons. Primer-dimers may be observed in NTCs, but should never occur in template-loaded samples if appropriate guidelines for minimizing primer complimentarity have been followed. In order to confirm that the product is in fact the intended specific amplicon, products should be analyzed by Southern blotting or DNA sequencing.
Laboratory tutorials
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Choice of primers: A number of considerations go into designing primers. Primers should not be self-complementary or complementary to each other, especially at their 3′ ends, to avoid primer dimers from forming. Keep the guanine and cytosine (G + C) content between 40% and 60%. Avoid long stretches of G + C, since they may form secondary structures. Addition of restriction enzyme sites to the 5′ end of the primers serves as useful vehicles for subsequent subcloning. Primer concentrations should be in excess of the template throughout the cycling. Typically, the primers are used over a 0.1–1.0 µM range. Lower concentrations may reduce artifacts and formation of primer dimers.
Experimental Protocols for Generation and Evaluation of Articular Cartilage
Published in Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi, Articular Cartilage, 2017
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi
While multiple chemistries are available for quantitative real-time PCR, SYBR Green and probe-based primer chemistries are the most common. SYBR Green binds to double-stranded DNA and has the advantage of requiring only forward and reverse primers, without needing a probe (reduced cost). However, significant primer optimization is required to prevent primer dimers and other false signals. Given the commercial availability of many primer-probe combinations, these are often the preferred choice.
Time-Dependent Effect of Graphene on the Microbial Activity of the Soil Under Single and Repeated Exposures
Published in Soil and Sediment Contamination: An International Journal, 2023
Wenjuan Liu, Yufeng Guo, Zihan Wang, Wenbo Deng
The V3-V4 region of the bacterial 16S rRNA genes was amplified using KAPA HiFi Hot Start Ready Mix (2×) (TaKaRa Bio Inc., Japan). The universal bacterial 16S rRNA gene amplicon PCR primers 341F/805 R (CCTACGGGNGGCWGCAG, GACTACHVGGGTATCTAATCC) was applied to amplify the V3-V4 region of bacterial 16S rRNA genes. The reaction was set up as follows: microbial DNA (10 ng/μL) 2 μL; amplicon PCR forward primer (10 μM) 1 μL; amplicon PCR reverse primer (10 μM) 1 μL; 2× KAPA HiFi Hot Start Ready Mix 15 μL (total 30 μL). The plate was sealed and PCR performed in a thermal instrument (Applied Biosystems 9700, USA) using the following program: 1 cycle of denaturing at 95°C for 3 min, first 5 cycles of denaturing at 95°C for 30 s, annealing at 45°C for 30 s, elongation at 72°C for 30 s, then 20 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s, elongation at 72°C for 30 s and a final extension at 72°C for 5 min. The PCR products were checked using electrophoresis in 1% (w/v) agarose gels in TBE buffer (Tris, boric acid, EDTA) stained with ethidium bromide (EB) and visualized under UV light. The AMPure XP beads were used to purify the free primers and primer dimer species in the amplicon product. Samples were delivered to Sangon BioTech (Shanghai) for library construction using universal Illumina adaptor and index. Before sequencing, the DNA concentration of each PCR product was determined using a Qubit® 2.0 Green double-stranded DNA assay and it was quality controlled using a bioanalyzer (Agilent 2100, USA). Depending on coverage needs, all libraries can be pooled for one run. The amplicons from each reaction mixture were pooled in equimolar ratios based on their concentration. Sequencing was performed using the Illumina MiSeq system (Illumina MiSeq, USA), according to the manufacturer’s instructions.