Application No. 229: PCR and real-time PCR experiments performed with DNA samples which have undergone multiple measurements in the UVette®, using the Eppendorf BioPhotometer plus
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.
This Application Note shows how four different primer sets were optimized to a single PCR protocol with the gradient function of the Mastercycler® pro from Eppendorf. The results also reveal the importance to perform a gradient run on any primer set in order to find out the primers’ workable temperature range and limitation.
DNA samples were measured multiple times in three different concentrations using the BioPhotometer plus and subsequently employed directly in real-time PCR and standard PCR experiments. These experiments did not detect any loss of quality of the DNA due to UV light as compared to unmeasured controls. Based on these results, we conclude that DNA samples which have been measured in the Eppendorf BioPhotometer may be used in suitable downstream applications.
This Application Note introduces a new protocol for multiplex PCR-based detection (VYOO, SIRS-Lab, Jena,
Germany) of bacterial and fungal pathogens. Directly from whole blood, sepsis-causing species are identified.
Sepsis is one of the most common causes of death in hospitalized patients whereas rapid pathogen detection is a
cornerstone in effective therapy.
DNA was isolated from EDTA whole blood samples of intensive care unit (ICU) patients suspected for sepsis. To
investigate these DNA isolates for the presence of sepsis-causing pathogens a microbial DNA enrichment was
performed followed by a standard multiplex PCR on the Mastercycler pro.
Species- and genus-specific primers for 34 bacterial and 6 fungal targets as well as 5 antibiotic resistances were
combined within two primer pools. The amplicons were analyzed by gel-based identification with custom-designed
DNA marker ladders. With this protocol, nucleic acid trace detection was successful with an overall sensitivity of
10 to 100 colony forming units (cfu)/mL whole blood.
In this study, the performance of the Eppendorf Mastercycler pro S and two thermocyclers of other manufacturers
were compared by detecting the PCR products in the log phase by means of fluorescence endpoint measurements.
The determined PCR performance and measured block homogeneity of the Eppendorf Mastercycler pro S
excelled in both aspects when compared to the competitive thermocyclers.
