STABILITY OF TAQ DNA POLYMERASE AT ELEVATED TEMPERATURES
Ampliqon Taq DNA Polymerase is a very stable enzyme with a half-life time of 40 minutes at 95°C. The half-life time rapidly increases with only a few degrees of temperature drop. To evaluate the thermostability we tested the activity of Taq polymerase at three different temperatures.
Ampliqon Taq DNA Polymerase of the same batch was distributed into several tubes and incubated at the temperatures 40 °C ± 2 °C, 25 °C ± 2 °C and 5 °C ± 3 °C. At specified time points (table 1) samples were collected in duplicates and transferred to -20 °C until analysis. One sample (in duplicates) was stored at -20 °C from the start to serve as T0 sample which never was exposed to elevated temperatures. The T0 sample serves as reference for 100 % activity and is used to prepare the standard series.
Table 1: Time points of Taq Stability Test.
Analysis was done in two ways. Firstly, real-time PCR was applied to monitor relative changes in enzyme activity. This was done by diluting the samples to a concentration, where the amount of Taq DNA Polymerase was limiting, thereby allowing to monitor changes in enzyme activity. Starting with the same dilution, a standard curve for Taq polymerase activity was prepared from the sample without incubation at elevated temperatures for relative quantitation. Secondly, an end-point PCR was performed to show the influence of enzyme activity to a standard PCR reaction. To challenge the system, the PCR was done using 3 dilutions of gDNA.
Ampliqon Taq Polymerase incubated at 40 °C:
Ampliqon Taq Polymerase incubated at 25 °C:
Ampliqon Taq Polymerase incubated at 5 °C:
AMPLIQON TAQ POLYMERASE IN THE FREEZE-THAW STRESS TEST
To test the resistance of Ampliqon Taq polymerase to freezing and thawing, a freeze-thaw test was performed applying 50 freeze-thaw cycles. To make the test even more challenging, Ampliqon Taq polymerase was kept in a buffer without glycerol. Glycerol is a cryoprotectant, and are normally part of the storage buffer where it serves to protect Taq polymerase during freezing conditions.
Samples were thawed at 30 °C for 7 minutes, shortly vortexed and spun down at room temperature (RT) and then placed on ice. 30 minutes after the start of thawing, samples were placed at -20 °C for at least 1 h before the cycle was started again. At defined numbers of cycles, the according samples were collected and stored at -20 °C until analysis. One sample was kept at -20 °C without any thawing to serve as reference for 100 % activity and to prepare the standard series.
Analysis was done using real-time PCR. Samples were diluted to a concentration, where the amount of Taq polymerase was limiting, thereby allowing to monitor changes in enzyme activity. Starting with the same dilution, a standard curve for Ampliqon Taq polymerase activity was prepared from the sample without freeze-thaw cycles. The standard curve amplification plots are shown in yellow for 100 % activity and in black for the rest of the standard series.
Ampliqon Taq DNA polymerase is a remarkable thermostable enzyme. Our results show that it is tolerant to incubation at elevated temperatures for long periods without losing activity: at 40 °C for 2 weeks; at 25 °C for 2 months and at 4 °C for 18 months.
Ampliqon Taq DNA polymerase can withstand a high number (> 40) of freeze-thaw cycles with only slight decrease in activity. At lower temperatures, Taq polymerase is stable for even longer time. Storage at -20 °C is therefore recommended in order to guarantee maximal shelf life. However, if you forget your enzyme on your lab bench even for several days, no harm is done due to the high stability of Taq polymerase at room temperature.
Taq DNA polymerase is mainly used to perform end-point PCR. In standard PCR setups, Taq DNA polymerase is present in vast excess, which makes the method tolerant to up to 50 % decrease of enzyme activity.
Ampliqon Taq DNA polymerase offers a perfect combination of heat resistance, robustness, specificity, sensitivity and yield and is commonly agreed to be one of the best polymerases available.