To evaluate the performances of Eppendorf 96-well twin.tec PCR plates, we carried out PCR reactions with twin.tec plates and PCR microplates 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 2.0 kb target. Further, there were also differences in temperature profiles between the two plates. In this article, we present evidence to substantiate that Eppendorf twin.tec plates have excellent temperature transfer characteristics and outperform competitor plates.
There are several suppliers of 96-well microplates for performing PCR reactions, and this format has become the most popular for high throughput research. One of the prime requirements for a good PCR microplate is the efficient transfer of block temperature to the reaction samplethis feature translates into a robust and reliable PCR reaction. Another desirable feature in a microplate is the mechanical sturdiness for unimpeded handling by robotic arm for automation. Eppendorf recently introduced the 96-well twin.tec plates to the life science research community with the goals of addressing the aforementioned requirements. Twin.tec plates represent a new generation of 96-well plates for PCR applications, which combines the advantages of two materialspolycarbonate and polypropylene. Polycarbonate is a rigid and stiff material and is therefore used to impart the required mechanical stability for the frame and plate surface before, during and after PCR. The walls of the wells are made of polypropylene, an ideal material for achieving rapid heat transfer from the block to the sample, thus leading to optimal results. Further, the manufacturing process and use of special virgin propylene provides thin-walled wells and a snug fit in the thermal cycler metal block, resulting in uninterrupted heat transfer from the block to the sample. The well-walls of twin.tec PCR plates are 20% thinner than the conventional thin-walled tubes and plates, and this translates into more efficient heat transfer to the sample as evidenced in the results.
In this article, we have made a comparative assessment on the performance of the Eppendorf 96-well twin.tec plate with a microplate from a commercial supplier. We amplified a 2.0 kb fragment from the beta globin region using both these plates. The gel analysis indicates that the amplified products are formed with better consistency in twin.tec plates. We also measured the temperature profile and present data that supports the superior heat transfer characteristics of Eppendorf 96-well twin.tec plates.
Materials and Methods
Genomic DNA from blood was isolated using an Eppendorf Perfect gDNA Blood Mini Kit. All PCR reactions were performed in four sets on the Eppendorf Mastercycler gradient thermal cycler** using human genomic DNA as a template. The primers were synthesized by Sigma Genosys.
The PCR target that was selected to compare the microplates was a 2.0 kb fragment from the human beta globin gene. It was amplified using Eppendorf Taq DNA polymerase, and the same protocols were followed for both twin.tec and competitor plates. All reactions were performed in four sets and were run on a 1% agarose gel for analysis.
For amplification, the following primers, reaction components and cycling conditions were used:
Forward primer: 5'-GAA GAG CCA AGG ACA GGT AC-3'
Reverse primer: 5'-CCT CCA AAT CAA GCC TCT AC-3'
Reaction components for PCR:
1.68 x PCR buffer (2.5 mM MgCl2 Final)
0.2 M of each primer
0.2 mM dNTPs
1.25 units Eppendorf Taq polymerase
50 ng human gDNA
MBGW to 25 l total reaction volume
Measurement of Temperature Profile During PCR Cycle
The Testo 945 measured the temperature profiles in the wells of microplates were measured during the course of PCR cycling (Manufacturer: Test, Inc. NJ, USA). This unit was calibrated and certified to NIST standards and is routinely used to calibrate cyclers in laboratories. The temperature measurements were carried out simultaneously in both the plates.
Our goal was to record the temperature profiles of both plates on the same instrument. To address this objective, the plates were cut in half and placed at the center of the thermal block. The high precision thermocouple probes were placed at the center of the wells, and the temperature was recorded following the start of a PCR cycle. The real time recording of the temperature profile was viewed using Testos Comsoft 3 software.
Results and Discussion
Comparison of efficiency of amplification
An Eppendorf twin.tec plate and a commercially available competitor PCR plate were evaluated for the amplification of 2.0 kb human beta globin fragment. The main criterion for this evaluation is efficiency of the plates for target amplification. Upon comparison of the yields of the product in gel electrophoresis (Fig. 1.), it was obvious that Eppendorf twin.tec plates performed well in terms of yield of PCR products. Note the samples shown in this analysis are from the same wells in both plates, D5D8, and were chosen as good representatives of the entire plateFigure 1: Amplification of 2.0 kb human beta globin target using Eppendorf 96-well twin.tec PCR plate compared to a commercially available competitor polypropylene microplate
Lanes 1 and 6: MWM (1 KB Ladder, Invitrogen)
Lanes 2-5: Competitor Polypropylene microplate
Lanes 7-10: Eppendorf twin.tec plate
The results demonstrate that the Eppendorf twin.tec PCR plate facilitates a highly efficient amplification of the 2.0 kb fragment with a consistent product yield in all the four reactions (Fig. 1). These results also show that the yield in the twin.tec plate is higher as evidenced by the higher intensity of the bands in all four wells. This observation prompted us to further examine any differences in temperature profiles in both plates.
Comparison of temperature profiles
The simultaneous sample temperature measurements during the course of the PCR cycle in both plates are shown in Figure 2. The green line represents the temperature trace of the Eppendorf twin.tec plate, while the red line represents the trace of the competitor plate. It is very clear from this figure that there are differences in the temperature attained by individual wells in both the microplates. An annealing temperature of 55.5 C was set for this experiment. Note that the red trace representing the competitor plate does not reach this temperature, while the green trace (twin.tec) does. Following annealing is the elongation step, for which a temperature of 72 C was selected. Again, the red trace does not reach this temperature, suggesting that a sample in that well would not be exposed to the set temperature. The same is also true for the denaturation step. We see a very clear pattern in the temperature transfer abilities between the twin.tec plate and the competitor plate. It is obvious that the twin.tec plates attained the desired temperature exactly in every step and every cycle of the PCR reaction, while the competitor plate always lagged behind. We can attribute this result to the fact that the walls of the wells of twin.tec plates are even thinner than the conventional thin-walled tubes, and this feature enables rapid heat transfer, ensuring more robust conditions for the PCR reaction and thus more reliable results.Figure 2. Temperature profile of Eppendorf twin.tec plate vs a competitor PCR plate.
When we take a closer look at a specific part of this graph, the difference in temperature transmission is even more obvious. An enlarged area of the annealing step is shown in Figure 3. A temperature difference of ~1 C can clearly be seen in the annealing step of the PCR reaction between the two plates. The samples in the competitors plate cannot attain the set temperature (55.5 C) because of this inefficient heat transfer. This difference in temperature transfer between the plates helps to explain the results in the previous experiment about the robust product formation with the twin.tec plate.
From the foregoing discussion, we can conclude that the ideal microplates
for PCR application are those that can effectively transfer heat to the
reaction mixture. All of the results from the above experiments point
towards this characteristic of twin.tec plates, which make them a perfect
choice for all temperature-sensitive applications
Eppendorf twin.tec plates offer many advantages over other PCR plates in the market. Primarily, twin.tec plates increase specific yields of some PCR products to a greater extent than the competing plates. This increase in yield is probably due to the excellent heat transfer properties of twin.tec plates, which are evidenced by the above temperature recordings. The superior performances of twin.tec PCR plates, combined with their structural rigidity, make them an excellent choice for all 96-well PCR applications.
* Practice of the patented polymerase chain reaction (PCR) process requires
** The Eppendorf Thermal Cycler is an Authorized Thermal Cycler and may be used with PCR licenses available from Applied Biosystems. Its use with Authorized Reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents.
Mastercycler gradient (U.S. Pat.6,210,958)