Hi Fernanda,

Is your negative control a sample with no RNA?

If so, maybe a second pair of primers can be helpful— I don't know how primer dimers can affect the signal, but it can be a possible source of false signals. Obviously you're working in a clean area, just make sure you're using filtered pipette tips as well.

Hello Fernanda Rufino da Silva

The contamination may be coming from

1) Your laboratory environment such as your pipettes, tips, hands, bench top and centrifuge.

To remove all possible environmental sources, when tracking down PCR contamination, you should use a 10% bleach solution to wipe down your bench top, pipettes, centrifuge, vortex, racks and thermocycler lid and buttons. Use unopened sterile PCR tubes and filter tips. Wear a dedicated apron, keep changing your gloves frequently and use equipment dedicated for qPCR. For instance, pipettes, centrifuge, and vortex you use when setting up a qPCR should be dedicated to qPCR setup.

2) Your reagents such as your polymerase, buffer, nucleotides, water, and other reagents.

You should check your reagents. You should systematically substitute each of your old reagents with a new (previously unopened) reagent and re-run your negative control. Whatever substitution(s) removes the contamination is the contaminated reagent, which should be discarded.

Other tips that you may consider.

1. You should set up a pre amplification and a post amplification area in your lab.

2. Keep all equipment and reagents separate.

3. Never have the amplified product in the pre amplification area.

4. Aliquot your reagents into smaller tubes and only work from one aliquot at a time. Not only will this help you deal with contamination, but it will also prolong the life of your reagents by reducing the number of freeze/thaw cycles.

5. Don’t flick your tubes open to minimize aerosols. It could contribute to your bench’s contamination. Instead take your time and use two hands to carefully open all PCR product tubes.

6. You should always set up your PCRs using master mixes made in the order as water, buffer, nucleotides, primers, polymerase, and finally, the template.

Good Luck!

Hi Hamid Kian Gaikani

Thanks for answering. Yes, my negative control is just water. My pipettes are also filtered. I will look into primer-dimers and try an experiment to see if they are related. Thank you for your time and help.

Hello Malcolm Nobre .

Thank you for your effort and help. You gave me a lot of great tips that I will put into consideration and practice.