I ran a full plate of qPCR standard yesterday and had no amplification. I was running a gradient on the plate to test the different annealing temperatures. When the ran finished it showed no amplification. I'm confident that I have all of my Primers and Probes as well as the DNA itself in my reaction mix. Also it's the same protocol that I've been using before so I'm confident that my ratios were correct. If all of my reagents were right what could it be? Thank you.
Confirm that the real-time PCR instrument is set at the correct wavelength to detect the right fluorophore.
Make sure you are not leaving out a necessary component from the reaction. Check the master mix components; it is possible that one of the components (such as the polymerase or probe/ primers) were mistakably left out. I recommend you to repeat the experiment once again to know if same problem persist. Confirm that your probes are not outdated or not working, by using another new one.
Also check that your cDNA conversion was successful or not (with spect. or running on gel).
If it is ok, change your dilution final volume and dilute it with lower volume.
If it is not ok, check your conversion process again.
Run the failed qPCR on an agarose gel to check for the amplicon.
To add to the above answer, make sure that you used the correct PCR settings on the real-time machine and that the program completed. If the power to the machine is interrupted, the run will automatically stop and will not continue.
Check the PCR products on a gel to see if nothing is amplified or if only the detecting didn't work.
If nothing is amplified, run the same reactions on another PCR intsrument (that could be a conventional "non-real-time" machine), to confirm that it's not a problem of your mixture. Run a well-established assay on the reat-time machine (can be a conventional assay - real-time detection is not required!) to confirm that the cyling in the real-time machine works (not).
If you have amplification but no detection, check the filter settings. Also run a well-established real-time assay to see if this gives you amplification curves. If not, there might be a problem with the light source, shutter or detector.
Hi Everyone. Thank you for your input but it looks like we found out our issue. I'll detail it here in case any of you run into the same problem. We are currently using the qTower product by analytikjena ( a Germany company). I confirmed that all of my settings were correct and all reagents were present and accurate. The issue , it turns out, is that a glitch in the software caused the sensor to stop working when I attempted to use a "temperature gradient" setting. If you run into a similar issue, (with similar equipment) you would want to look out for no reads when your machine scans. Usually, if it's a reagent related problem, you would see something going on with the different data points as they're collected. In this case, no data points were being collected despite the machine going through all of the different steps. In this case, it would be best to reach out to the vendor with a saved file of the run that failed so they can diagnose the issue.
Thanks again for your input all.
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