Hi, I have NTC contamination in real-time PCR. I checked all suspicious items such as primers, master mix, and distilled water, and also the workspace. but unfortunately, I can not remove the contamination.
It onlt takes a tiny amount of contamination as every 10 cycles of pcr is 1000 times more product. Try dismantling and thoroughly cleaning all of your pipettes and set up a pcr with another researchers work area,pipettes,reagents (except your primers) then introduce your reagents one at a time.
A better way is to design 2 new primers sitting outside (larger pcr product) of your contaminated primer set then any contamination cannot amplify and try to be much more careful opening plates and tubes and making sure that nothing moves from the post pcr lab area to your pce setup area especially pipettes
Try asking someone else perform it for you.
Have you tried using complete new set up of reagents
Hi, If you check any of the items and contamination is not evident, it occurred during the sampling process of your work.
For this, it is better to first place the column related to NTC with a distance from other columns and first add the corresponding Master in the corresponding strip and then add the Master in other columns and strips. In fact, try to strip NTC a distance in both horizontal and vertical mode with other strips that contain samples containing DNA and cDNA &... is to be. Also, if the strips are connected to each other in the form of columns and 8 pieces, do not cut the columns in any way.
Also, to prevent contamination, it is better to perform all the steps under the biological hood and check the materials and equipment once more and make sure. And the work tools must be completely sterilized and UV lighted before work.
Of course, all these conditions that I mentioned are for when you are sure that your materials are not contaminated.
Hi Zeynab,
For such issues, you can simply contact Dr. Shanehbandi at Tabriz Medical University, an expert in PCR and troubleshooting. 09144160144
All the best,
Daniel
Sometimes the only way to remove the contamination is to design new primers that are "outside" the original amplified region.
The other recommendations are:
- Check that it's really contamination as opposed to unused primers by running out some of the sample on an agarose gel.
- Throw away ALL of the reagents. I mean all of them - water, primers, buffer, RT-mix. Start over with all new reagents.
- Never use the same micropipettes to set up PCR that you use to handle amplified PCR products. Don't use the same bench area either.
And remember that the goal is to get the qPCR working properly, not to chase down the exact source of the contamination.
Good luck!
A few more ideas that might help
If you have filter tip pipets those can help with keeping the pipets free from aerosol contamination.
Make sure your tip collection (waste container) is far away from your actual PCR tubes so you're not ejecting tips close to the sample setup.
Have your own aliquots of all the reagents, templates, primers, water and don't share! I had contamination one time because a lab mate was helping themselves to my sterile nuclease-free water using non-filter tips and the pipet they used for genomic DNA extraction.
good luck!
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