To evaluate the performances of eppendorf 96-well twin.tec PCR Plates we carried out PCR reactions with twin.tec plates and PCR micro-plates from another commercial microplate supplier and compared the reaction products by agarose gel analysis. Additionally, simultaneous temperature profiles were recorded in both plates using dual temperature probes during the course of a PCR reaction.
As a result of these experiments, we found differences in efficiencies of the amplification of a 2 kb target. Further, there were also differences in temperature profiles between the two plates.
This Application Note provides a comparative overview of real-time PCR reactions at 20 μl, 10 μl and 5 μl reaction volumes, each generated on the Mastercycler ep realplex real-time PCR system. SYBR® Green I- as well asTaqMan® probe-based assays were performed, and the reaction setup was done with the aid of the epMotion® 5070 automated pipetting system.
It is shown that real-time PCR reactions with small reaction volumes can successfully be performed on the Mastercycler ep realplex: The reaction efficiencies obtained for the presented assays, including the lowest volume reactions, were comparably good and the Ct values obtained from the 10 μl- and 5 μl- reactions were only slightly higher than those detected for the 20 μl setups.
The new heated lid technology “vapo.protect”, featured by the Mastercycler pro, achieves improved evaporation protection even at the corner and edge positions of the thermo block. The tighter sealing of the newly developed fluid filled cushion incorporated in the heated lid, leads to an improved evaporation protection. With its demonstrated minimization of technical variance on PCR, the Mastercycler pro makes a significant contribution to higher comparability and reproducibility.
This application note provides a streamlined protocol for DNA sequencing using small reaction volumes on the Mastercycler® pro. We performed PCR in reaction volumes of 10 μl, 20 μl and 50 μl and compared the obtained PCR product concentration. Furthermore, sequencing reactions were carried out in 5 μl, 10 μl and 20 μl and the quality of the obtained sequencing data was evaluated.
We show that PCR and sequencing reactions with small reaction volumes can be performed successfully on the
Mastercycler pro: The obtained concentration of the PCR products and the quality of the obtained sequences,
including the lowest volume reactions, were comparable.
Robustness of the method was proven by sequencing the entire coding sequence of the human IRS1 gene in 95
bladder tumor samples using the smallest tested volumes. Using this protocol, sample and reagent consumption
was considerably reduced.
The influence of various consumables on the results of real-time PCR is described. In comparison to transparent micro test tubes, the white wells of the Eppendorf® twin.tec real-time PCR plates result in a clear amplification of the fluorescence signals and a reduced influence of the thermoblock on the reflection of the signal. These effects in turn lead to increased reproducibility and improved sensitivity in real-time PCR experiments.
This application note demonstrates that the reaction volume in real-time PCR experiments can be significantly reduced by using Eppendorf® twin.tec real-time PCR plates. These plates enable multiplex analyses to be performed with a total volume of 5 µl without loss of sensitivity or reproducibility. Reducing the reaction volume can considerably cut costs in real-time PCR.
The certified Eppendorf Biopur standard has been created to ensure the highest possible purity of laboratory consumables for applications in cell culture, IVF, diagnostics and other areas where protection against contamination is crucial. In this report, we present background information about parameters that are of critical importance for the purity of these consumables.
