30X, 72 (5 min). Reaction conditions: 1X PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl, 1.5 mM MgCl2), Primers (0.5 ), polydT primer (1 ), 0.16 mM dNTPs, 0-0.25 mg Human brain total RNA, 25 U reverse transcriptase, 0.3 U Taq DNA polymerase, 25 mL. Figure 8: Evaluation of CleanAmpTM Primers in onestep reverse-transcription PCR using SuperScriptII Reverse Transcriptase (Invitrogen) and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen). For the gene of interest, the PCR primers were unmodified, contained CleanAmpTM Turbo modification or contained CleanAmpTM Precision modification. Reverse transcription utilized a polydT18 primer. Reactions contained Taq DNA polymerase and SuperScriptII or M-MLV Reverse Transcriptase.
or DNA polymerase(15) mediated extension of primers to form primer-dimer and/or non-specific products at the less stringent temperatures of reverse transcription. To improve the sensitivity and specificity of RT-PCR, inhibition of such primer extension at lower temperatures is required. One approach to improving the specificity of one-step RT-PCR is to employ CleanAmpTM Primers. By introducing a CleanAmpTM Primer pair, only the RT primer can elongate during reverse transcription. This reduces lower-temperature, non-specific amplicon formation from extension of PCR primers. At higher temperature, the CleanAmpTM Primers are activated, allowing for greater specificity of primer extension during PCR. CleanAmpTM Primers provide a solution to non-specific amplifications and also enable other more universal RT priming methods for applications such as multiplex one-step RT-PCR. CleanAmpTM Primers improve sensitivity and specificity regardless of reverse transcriptase (RT) enzyme CleanAmpTM Primers decrease competing PCR primer extension during the RT step with a number of commonly used RT enzymes.1448428-04-3 manufacturer To illustrate this, PCR primer sequences were prepared as either a) standard, unmodified primers or b) one of two types of CleanAmpTM Primers (16,17).635702-64-6 manufacturer CleanAmpTM Turbo Primers
and slower-releasing CleanAmpTM Precision Primers differ in the rate of temperatureinduced formation of the corresponding unmodified primer. The following RT enzymes were tested: SuperScriptII Reverse Transcriptase (Invitrogen) and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) (Figure 8), with similar results found for both RT enzymes. The use of unmodified PCR primers resulted in formation of several non-specific amplicons, with the desired amplicon (264 bp) being formed at low relative abundance. Amplicon formation was enriched when CleanAmpTM Turbo Primers were used. In the case of CleanAmpTM Precision Primers, amplicon formation was also enriched to a slightly lesser degree. However, reactions containing Precision Primers displayed improved specificity.PMID:28723037 CleanAmpTM Primers can also be utilized with reactions employing either total RNA or poly(A)+ RNA. Experiments have shown that with various RNA tissue sources, one step RT-PCR displayed improved specificity when CleanAmpTM Primers were employed. Furthermore, one-step RT-PCR experiments have been evaluated in Real-Time qRT-PCR using CleanAmpTM Turbo and Precision Primers. The use of CleanAmpTM Primers enhanced the specificity of the reaction compared to the unmodified primers. CleanAmpTM Turbo Primers displayed the most significant increase in amplicon formation. Overall, CleanAmpTM Primers demonstrated marked improvement of sensitivity and specificity in one-step en.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