contamination is a huge problem.10 cycles of pcr gives 1000 times more product so even microscopic amounts become visible and take over the reaction. Clean up your pipettes inside and out with bleach. Clean your working area thoroughly with bleach and use new tubes/strips/plates from an area not near to your working area which is possibly contaminated. Get a fresh lab coat and only open pcr tubes/strips in the electrophoresis area not near your pcr setup areaand always run pcr product gels away from the pcr setup area. short term borrow reagents and pipettes and set up pcr in other peoples areas. The only thing that works for certain is to redesign your primers with at least one primer outside of your contaminated primers then the contamination cannot amplify but you have to clean up your working practices or even this solution will eventually become contaminated

contamination is a huge problem.10 cycles of pcr gives 1000 times more product so even microscopic amounts become visible and take over the reaction. Clean up your pipettes inside and out with bleach. Clean your working area thoroughly with bleach and use new tubes/strips/plates from an area not near to your working area which is possibly contaminated. Get a fresh lab coat and only open pcr tubes/strips in the electrophoresis area not near your pcr setup areaand always run pcr product gels away from the pcr setup area. short term borrow reagents and pipettes and set up pcr in other peoples areas. The only thing that works for certain is to redesign your primers with at least one primer outside of your contaminated primers then the contamination cannot amplify but you have to clean up your working practices or even this solution will eventually become contaminated

Thanks Dr Paul, in fact I changed the lab coat and changed the layout design by bringing all the NTC towards corner of the plate. I dispensed the reagents first to all NTC and closed them immediately with piece cover (sealer which we use to seal at the end) before I dispense to other wells. After pipetting the DNA, I opened the NTCs and closed whole plate carefully. But still I got amplification in NTC, so crazzy...

what I am suggesting is that when and where the NTC samples are makes no difference. Peeling back pcr seals after the pcr reaction an produce a spray of droplets which spread over the working area and dry out.Then dust has post pcr product on it so even the slight breeze of people moving around the area or air conditioning can spread contaminated dust into reagents or plates so contamination can get into some, or all samples and even the equivalent of 1/1000 of one ul as contamination amplified over 30 cycles would (mathematically) produce enough pcr product equivalent to about a litre of pcr product. Sometimes even the static electricity on plastic plates can attract dust which may carry contamination.

what I have said so far assumes post pcr contamination which shows up at low cycle numbers, 34 cycles is a huge amount of amplification and rtpcr very sensitive so in your case I suggest that the contamination is from native dna in one of the reagents. SYBR is simple and non biological so possibly your water is contaminated with a bug whose genome is amplifiable by your primers . Years ago it sometimes happened that the taq contained some dna from its parent organism and this might amplify with some primers but things improve with time. I assume that the product is not primer dimer. Sequencing the ntc product may give a clue to the answer

I think Paul has some good thoughts. A few things I discovered from experience.

1) Some qPCR cyclers will show you what temp the primer annealed (some cyclers you need to turn on this feature or add it to your qPCR protocol). If the temp for the NTC annealing and the temp for the actual product annealing are different, it is some kind of contamination not from your samples (not over-spray or accidental mixing up of samples, more likely to be contaminated reagents or DNA/RNA from another source such as you).

2) Two unlikely sources of contamination we found were our water and the primers/SYBR reagent themselves. For the water, we bought new bottles of DNA/RNA-free molecular or PCR grade water and made small aliquots that we only use once then throw away any remainder. Similarly, we aliquot the SYBR and primers to reduce the likelihood of contamination of the stock reagents.

3) Over-spray from pipetting or other aerosols are a huge potential source of contamination. Clean pipettes and filter tips are a bare minimum. I also advise my students not to push past the first stop on the pipette (a little reagent or RNA will remain in the tip, but that is better than the over-spray that is produced when you expel the last tiny bit from the pipette. Last, we have tried a variety of ways of covering the wells and the best bet is something plastic (like a pipet box lid) that can be sterilized with ethanol or bleach. Keep the rows you are not pipetting covered as best you can.

4) Try a DNAse treatment of your samples either during the RNA purification or after.

5) If amplification in your NTC occurs after cycle 38, or >10 cycles beyond your samples (example samples = 21 and NTCs = 31), you can assume it is not real and caused by some sort of contamination. Some would argue you can ignore this and the NTCs are in fact negative, but I would make such assumptions with caution.

Good luck!