When using Turbo Primers (Figure 6A), with Turbo Primers efficiently detecting 50 copies of Lambda genomic DNA, where unmodified primers could only detect 5,000
Figure 6: Multiplex reactions over a wide range of template amounts. A. Performance of Turbo and unmodified primers with a varying amount of template. B. Quantitative analysis of amplicon yield and primer dimer formation for experiment performed in Figure 6A. For each template concentration, amplicon yield was normalized to Turbo.
copies. Furthermore, the longer targets appeared to amplify less efficiently than the shorter targets when using unmodified primers, with the longest 962 bp amplicon not forming until 50,000 copies of template were employed.1809249-37-3 Synonym On the other hand, at all template concentrations examined, Turbo Primers amplified all three targets with similar efficiency. Furthermore the use of CleanAmpTMPrimers improved PCR performance by reducing primer dimer formation (Figure 6B). In summary, efficient amplification by Turbo Primers is less restricted by target size limitations, all three amplicons being formed over a broad range of input template concentrations at increased sensitivity. CleanAmpTM Turbo Primers outperform in real-time multiplex PCR To confirm that the range of detection was also reproducible in quantitative real-time PCR, a duplex reaction using Taqmanprobe detection was performed. Much like the endpoint experiments, at low template concentrations, the detection of amplification is much more sensitive using CleanAmpTM Turbo Primers. In this duplex reaction, the difference in Cq between unmodified and Turbo Primers increased as 3
template concentration decreased (Figure 7). In some cases, such as the in the L600 target, no Cq is observed at 50 copies for the unmodified primers.150408-73-4 site Turbo Primers provide earlier detection of successful amplification, whereas amplicon detection with unmodified primers is delayed or not present. Overall, the use of CleanAmpTM Turbo Primers in multiplex PCR provides several advantages, which include greater amplicon yield and lower primer dimer formation. Turbo Primers provide great flexibility in assay design, as a wide range of primer concentrations produce robust, nonpreferential amplification. Furthermore, greater sensitivity is achieved for both endpoint and real time assays, with a 100fold increase in the limit of detection. one-step rt-pcr applications With the advent of microarrays, the need to validate the massive amount of gene expression results has grown significantly. Reverse transcription PCR (RT-PCR) has become the gold standard for validation of microarray gene expression profiles(8,9).PMID:30000266 The typical RT-PCR reaction consists of a two-step protocol that involves a lower temperature reverse transcription step followed by an elevated temperature PCR step(10). The extra manipulation procedures inherent to a two-step protocol can introduce opportunities for contamination. A one-step RT-PCR protocol provides a streamlined, high-throughput technique that reduces the chances of contamination(11). Another advantage for a one-step protocol is that replicates will repeat both the reverse transcription and the PCR step. However, one-step RT-PCR is not without its own inherent problems. In many cases one-step RT-PCR reactions are not as sensitive as two-step(12,13). The lack of sensitivity is likely a result of reverse transcriptase(14) 4
Thermal cycling conditions: 42 (30 min), 95 (10 min), [95 (30 sec), 60 (30 sec), 72 (30 sec)].MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com
