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PCR Cycler Validation - Frequently Asked Questions
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| Q: |
What’s the definition of accuracy and spread? |
| A: |
Accuracy is defined as the difference in display and actual block temperature. Spread (or in-uniformity) is the difference in temperature between the wells of a cycler block. |
| Q: |
Why should I test my PCR thermal cycler? My results are always OK… |
| A: |
It is very important to know how your thermal cycler behaves. Negative results could be caused by temperature differences in the block, and could be positive in reality. Biological controls are not sufficient to compensate for these differences. |
| Q: |
Can these temperature deviations really influence my PCR results? |
| A: |
Yes, when the denaturation temperature is too high, the DNA polymerase will soon loose activity during the PCR process, resulting in low or no yields. Annealing temperatures are very specifically determined. Changes will result in specific bands or no annealing at all. |
| Q: |
What’s an overshoot and an undershoot? |
| A: |
Due to a lack of control, cyclers often are not able to reach a target temperature in a stable way. This means that a denaturation temperature can go much higher than the set temperature, before reaching its plateau. For the annealing temperature plateau, undershoots of up to several degrees Celsius are not uncommon. |
| Q: |
Isn’t it possible to do a temperature measurement myself? |
| A: |
It will be very difficult. CYCLERtest uses the MTAS (Mobile Temperature Acquisition System) that was developed by GENOtronics for more than 5 years. It uses very sophisticated temperature sensors (thermistors), electronics and software algorithms, to correct for possible deviations. The overall accuracy of this system is better than 0.1°C. It also measures multichannel and dynamically. You or your Technical Services Department might be interested to sublicense an MTAS system to measure all thermal cyclers in your institute or company. |
| Q: |
Which PCR thermal cycler is the best? You should know, because you measure many different brands… |
| A: |
Despite all the data that we have available, CYCLERtest will refrain from making any statements on this issue. First of all, we want to be an independent measurement institute, and not a PCR apparatus consultant. Secondly, it is very difficult to make general statements about certain brands or types, because there are already huge differences between machines of the same type or brand, and also the age of a cycler is of influence. |
| Q: |
What if you do a performance test on my PCR cycler, and it turns to be out of specs? |
| A: |
You have several options. In case the cycler is still within warranty, we’d suggest that you go back to your supplier, and ask them to take action. Some brands of cyclers can be re-calibrated back into specifications. When your cycler is out of warranty it might be necessary to repair (if possible) or replace it. The last option is to take into account the measured differences (in accuracy, spread, over and undershoots) when using the machine and when developing a protocol. |
| Q: |
How long does a CYCLERtest take? |
| A: |
Approximately 45 minutes per block. We can do the performance testing at your location. |
| Q: |
What’s the influence of the disposable on the temperature performance of a PCR cycler? |
| A: |
The mass of the tubes, strips or plate and its contents influence the performance of a PCR cycler, depending on its control capabilities. We can also perform half or full load tests to visualize these effects. |
| Q: |
Can you test all brands of thermal cyclers? |
| A: |
Yes, as long as it has a regular 0.2 ml, 0.5 ml or 384 well block. We can also test gradient cyclers with our special gradient protocol, and multiple and/or exchangeable blocks. |
| Q: |
What does the word validation mean? |
| A: |
We test your PCR cycler against internationally accepted temperature standards, and the measurement system itself is complying with the official temperature measurements regulations. This means that if you have your PCR cycler performance tested with our system, it is also validated according to these regulations. |
| Q: |
How often do you advice to have my PCR cycler validated? |
| A: |
We advice to have a validation test at least every year, or every 250 runs, whatever comes first. Some laboratory, national or international regulations might require you to do this more often. |
| Q: |
What’s the warranty on your validation and performance test? |
| A: |
There is no “warranty” on the test. We show the output performance of your cycler at the time of measurement. The performance measurement cannot predict what will happen with the accuracy and spread of your cycler in time, nor will it predict if your cycler breaks down the next day. Machines, so also PCR apparatus wear in time, so will their performance. |
| Q: |
What if I want to check my cyclers’ performance more often? |
| A: |
We have developed the so-called DriftCon system to check the performance of the cycler weekly or daily. It is an enduser temperature measurement fixture that will show the drift of the accuracy and the spread of your PCR cycler in time, and in relation with the global field specs of that brand and type of cycler. |
| Q: |
Why does my cycler almost never comply to manufacturers’ specs? |
| A: |
Manufacters’ specs are marketing tools. They are almost always set too high, so the chances that your cycler is within these specs are minimal. It is much more realistic to work with so-called field-specs: real life specs derived from our global database of brands and types of thermal cyclers. |
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