qPCR with SYBR® Green .
Our primaQUANT CYBR qPCR Master Mixes with SYBR® Green are extremely fast. Due to their high sensitivity (detection limit < 1 pg) they provide reproducible results even under difficult conditions.
The primaQUANT CYBR Master Mixes are very temperature stable. They can be pipetted uncooled and can be stored for many months without any problems.
For perfect visual control during pipetting, primaQUANT CYBR qPCR Master Mixes are dyed blue as standard. The dye does not affect performance, nor is a change to the protocol necessary.
Hard Facts .
With the primaQUANT CYBR qPCR Master Mix, you can perform genotyping using High-Resolution Melting Curves (HRM) – without the need for agarose gels.
Li, W., Matsuoka, M., Kai, M., Thapa, P., Khadge, S., Hagge, D. A., Brennan, P. J., & Vissa, V. (2012). Real-Time PCR and High-Resolution Melt Analysis for Rapid Detection of Mycobacterium leprae Drug Resistance Mutations and Strain Types. Journal of Clinical Microbiology, 50(3), 742-753. https://doi.org/10.1128/JCM.05183-11
Słomka, M., Sobalska-Kwapis, M., Wachulec, M., Bartosz, G., & Strapagiel, D. (2017). High Resolution Melting (HRM) for High-Throughput Genotyping-Limitations and Caveats in Practical Case Studies. International Journal of Molecular Sciences, 18(11), 2316. https://doi.org/10.3390/ijms18112316
Lefever, S., Rihani, A., Van der Meulen, J., Pattyn, F., Van Maerken, T., Van Dorpe, J., Hellemans, J., & Vandesompele, J. (2019). Cost-effective and robust genotyping using double-mismatch allele-specific quantitative PCR. Scientific Reports, 9(1), 2150. https://doi.org/10.1038/s41598-019-38581-z
Single nucleotide polymorphism (SNP) genotyping is widely used in genetic studies to determine the factors underlying inherited traits.
Baris, I., Etlik, O., Koksal, V., Ocak, Z., & Baris, S. T. (2013). SYBR green dye-based probe-free SNP genotyping: introduction of T-Plex real-time PCR assay. Analytical Biochemistry, 441(2), 225-231. https://doi.org/10.1016/j.ab.2013.07.007
Dhas, D. B. B., Ashmi, A. H., Bhat, B. V., Parija, S. C., & Banupriya, N. (2015). Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR. The Indian Journal of Medical Research, 142(5), 555-562. https://doi.org/10.4103/0971-5916.171282
Pang, Y., Zhou, Y., Wang, S., Lu, J., Lu, B., He, G., Wang, L., & Zhao, Y. (2011). A novel method based on high resolution melting (HRM) analysis for MIRU-VNTR genotyping of Mycobacterium tuberculosis. Journal of Microbiological Methods, 86(3), 291-297. https://doi.org/10.1016/j.mimet.2011.05.016
Bai, L., Deng, Y.-M., Dodds, A. J., Milliken, S., Moore, J., & Ma, D. D. F. (2006). A SYBR green-based real-time PCR method for detection of haemopoietic chimerism in allogeneic haemopoietic stem cell transplant recipients. European Journal of Haematology, 77(5), 425-431. https://doi.org/10.1111/j.1600-0609.2006.00729.x
Weksberg, R., Hughes, S., Moldovan, L., Bassett, A. S., Chow, E. W., & Squire, J. A. (2005). A method for accurate detection of genomic microdeletions using real-time quantitative PCR. BMC Genomics, 6, 180. https://doi.org/10.1186/1471-2164-6-180
To determine the expression of a gene (gene or RNA expression), the total RNA must first be transcribed into cDNA.
For the separate cDNA synthesis is our primaREVERSE RT Kit the first choice.
Alternatively, you can also use our primaQUANT 1STEP One-Step RT-qPCR Kit and directly use RNA in a qPCR.
The length of telomeres is well suited as a biomarker for cell aging. This also allows studying the impact of environmental influences on cell aging processes. qPCR is a rapid, reliable, and inexpensive method to study telomere lengths.
Lin, J., Smith, D. L., Esteves, K., & Drury, S. (2019). Telomere length measurement by qPCR – Summary of critical factors and recommendations for assay design. Psychoneuroendocrinology, 99, 271-278. https://doi.org/10.1016/j.psyneuen.2018.10.005
High Resultion Melting Curve Analysis (HRM) makes it easy to detect the smallest changes in the genome, such as SNPs and mutations, using qPCR.
Matsuda, K. (2017). PCR-Based Detection Methods for Single-Nucleotide Polymorphism or Mutation: Real-Time PCR and Its Substantial Contribution Toward Technological Refinement. Advances in Clinical Chemistry, 80, 45-72. https://doi.org/10.1016/bs.acc.2016.11.002
Simko, I. (2016). High-Resolution DNA Melting Analysis in Plant Research. Trends in Plant Science, 21(6), 528-537. https://doi.org/10.1016/j.tplants.2016.01.004
Mohammad Rahimi, H., Pourhosseingholi, M. A., Yadegar, A., Mirjalali, H., & Zali, M. R. (2019). High-resolution melt curve analysis: A real-time based multipurpose approach for diagnosis and epidemiological investigations of parasitic infections. Comparative Immunology, Microbiology and Infectious Diseases, 67, 101364. https://doi.org/10.1016/j.cimid.2019.101364
Next-generation sequencing requires accurate quantification of the DNA library to be sequenced for optimal results and high yields.
Such DNA libraries can be quantified inexpensively, rapidly and accurately by qPCR. By avoiding unnecessarily long PCR amplifications, the NGS workflow can be optimized, and amplification biases are also minimized.
Buehler, B., Hogrefe, H. H., Scott, G., Ravi, H., Pabón-Peña, C., O’Brien, S., Formosa, R., & Happe, S. (2010). Rapid quantification of DNA libraries for next-generation sequencing. Methods (San Diego, Calif.), 50(4), p15-18. https://doi.org/10.1016/j.ymeth.2010.01.004
Copy number variants can be measured by SYBR-Green qPCR using different Cq values after a calibration. In addition, the melting curve information allows an estimation of whether there are SNPs within the CNVs.
Loewe, R. P. (2013). Combinational usage of next generation sequencing and qPCR for the analysis of tumor samples. Methods (San Diego, Calif.), 59(1), 126-131. https://doi.org/10.1016/j.ymeth.2012.11.002
Diagnosis of pathogens can also be performed by nucleic acid analysis.
DNA pathogens (DNA viruses, bacteria, fungi) can be analyzed directly by quantitative PCR. For RNA pathogens (RNA viruses, e.g. SARS-COV2), a transcription of RNA into cDNA is necessary. For this purpose, we offer the primaREVERSE RT kit, a cDNA synthesis kit. Alternatively, you can perform cDNA synthesis and qPCR in one step with our primaQUANT 1-STEP Master Mixes.
Due to the particularly high purity of our primaQUANT Master Mixes, they can be used for quality control, e.g. to exclude the possibility that a product is contaminated with nucleic acids or nucleases.
For all qPCR cyclers .
Our qPCR 2x Master Mixes are suitable for all common qPCR cyclers and optionally available with ROX.
We have compiled a table for you showing whether your device uses ROX(6-carboxy- X-rhodamine) for normalization and which concentration, if any, is required.
2x SYBR® Green Master Mixes .
Available with ROX or without ROX – suitable for all qPCR instruments.