Hello everyone, I am working on Real time PCR assay. We set up 2-3 PCR assays daily. Though the clean and controls are handled in different biosafety hoods we sometime get internal control amplification in NTC ( no templet control). Which chemical reagents should be used for fogging to remove aerosol contamination. Our wells containing only PCR master-mix and primer probe shows no amplification of internal control which negates the possibility of contaminated reagents.
Hi Mandar H Rasane , if you are working inside a biosafety hood, the downward draft of the laminar air flow should prevent any aerosol contamination. In this case I feel contaminated nuclease-free water might be responsible for the signal in your NTC. Let the air flow run for good few minutes before you start to work and as you always do, spray with generous amounts of 70% IMS/ethanol.
You can also try to run a second set of NTC in a different PCR plate alone with no other samples. This will tell you if there is a problem of cross contamination. There is a good chance that microdroplets from adjacent sample well contaminated the NTC before you sealed the plate.
I suggest not to use any fogging as most techniques create particulate matter which will block your filters resulting in inadequate filtration and more chances of contamination. It is a good idea to check when was the filter last replaced in your current biosafety hood.
Ritihaas Surya Challapalli Thank you for the suggestion.
We have changed NFW lots and also tried sterile water for injection procured from medicine shop. but there was no success. WE have set up reaction in different hood as well as outside hood on a simple bench top but again the NTCs are showing IC amplification. We have tried 6% Hypochlorite spray but the results are not consistent, ie out of 8 NTC we get 5/6 clean.
We use 8 well strip of 0.2 ml Tube and not adding any sample since we observe the contamination. we just set up NTC.
what else do you suggest
At this point nothing else comes to mind other than checking the calibration on your PCR machine. Did you try to run calibration standards to validate the machine?
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