Hi RG community!
I'm writing this topic because one of my friends told me he has been experiencing evaporation issues with the Applied Biosystems' 9700, Veriti and ProFlex Thermal cyclers he has every time he performs an end-point PCR with 0.2 mL PCR tubes. This issue remains even when using Applied Biosystems PCR tubes, actually, it seems to be worse with the PCR tubes from this manufacturer.
The only way to solve this issue has been using mineral oil, which, as you may know, it's not too practical when you have to perform too many reactions.
He told me that ten years ago he used some tubes from another manufacturer that didn't have this problem and didn't require him to use mineral oil. At that time he was able to perform 10 uL reactions without experiencing any kind of evaporation in the 30-40 tubes he used. Nowadays that seems to be impossible even though he has domed-cap tubes. As mentioned, the only way he has found to avoid that issue is by using mineral oil.
Could you please help me with some suggestion that allows him to perform his PCR reactions without using mineral oil to avoid evaporation? I mean, some PCR tubes that work as they should or something that guarantee there won't be any sort of evaporation when performing the PCR.
I hope you can help me.
Best regards,
Ivan.
Iván León
Looks like the problem is with the lid temperature of the PCR machine it's has nothing to with the tubes if the tube caps have been secured firmly before running the reaction. Run a calibration and check if the lid temperature. The temperature of the lid should be higher than the temperature of your reaction tube to prevent evaporation and condensation on the lid.
My suggestion is, check the lid temperature of the PCR. Even if the PCR digital board shows 105 C, do run calibration and check if it actually heating up to 105 C
Cheers!
As suggested earlier, it may be related to lid temperature definition and actual temperature inside the hot lid. Certainly, the quality of the tubes also come into play. To rule out it, you can try with tubes provided by different manufacturer. . Going for mineral oil overlay is not a good option at this moment. You have to increase the volume further for proper amplification, if you have downstream process.
You can use inbuilt troubleshooting. Then, run your PCR with water alone to check evaporation. Concentrate in all four corners and center to ensure uniform heat distribution across the format. If its not satisfactory, go for actual calibration provided by the local technical support team, ABI.
The tubes with flat lids tend to get loosen with PCR lid heat on, However, if you use the tubes with domed shaped lids, you should not have this problem.
Sometimes the lid does tightly touch the lid, this will have the same issue as you have. The pad transfer the heat from the lid to the tube cap.
This might be because of higher lid temperature. Kindly get the instrument checked and calibrated. Also, try running the PCR with double the quantity of reaction mix.
Junjie Shao, can you recommend any specific product? I've been trying to find something, but the only thing I found was a pad from Bio-Rad (#ADR5001) that for some reason has a magnet on one side, and some other pads that are specific for qPCR and so have holes over every well. Thanks in advance!
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