Hi, did you always use it once (the water, prrimer,...)? We did everything with reagents in Eppis (the autoclaved water was in single Eppis etc.). We used it only one time! We made aliquots from every single reagent (not Tay Polymerase). Cheers, Nadine

Are you sure your (c)DNA (?) is good? Have you confirmed good fit of the primers with alternative methods (end-point PCR)?

@Marcin Nowicki : The question was why there is an amplification at Ct 31-35 in the NTC non-template control, not the amplification efficiency!

Nadine A .Gund @ thanks for your reply. Yes, I always make aliquots of every single reagent like primers, water, SYBR green master mix. But after taking all precautions, still getting amplification in NTC. 

Marcin Nowicki @ thanks for response. Ya, I am absolutely sure for my cDNA and anyway I am not using cDNA in negative control. So there is no mean of good or bad cDNA. My problem is amplification in negative control.

@ Sabine Strehl, you are absolutely right.

How often did you have amplification in your NTC? Did you repeat the experiment? Cheers, Nadine

@Nadine, I am getting amplification everytime. Yes, I repeated my every experiment  twice.

@ Roopam Sharma, yes, i  have checked my PCR product on agarose check and it is not primer dimer.. proper amplicon size band in NTC and however, the intensity is less as compare to the sample.

After so much trial 3 possible reasons are still in hand

1. Primer dimers still in question, usually 80-140 bp and have sometimes exactly the same size with amplicon. Hot start and/or more stringent conditions can be tried to get rid of that dimers.

2. Carry over contamination can be still on subjection. DNAse treatment excluding primers may be tried for NTC.

3.  This is PCR and only pussyCat knows the reason. If you have excess time, Stop the experiments for at least one week.  Repeat with the same conditions. If you get rid of the band do not spend time to find the reason. If still getting the same band apply item 1 or 2.  

One way to check if you are obtaining primer dimers  is to perform a melting curve analysis after running the PCR. Usually primer dimers has a lower Tm than your specific amplicon.

I guess you are using a SYBR green based method? NTC at CT 31-34: for some primers we get this as well, but for others not. If that´s the case for you, then probably it´s primer dimers, and as someone wrote already - primer dimers can generate a product of similar melting temperature/size as your specific product.

More importantly, before investing too much time in trouble shooting something that is not a trouble check this: at which CT does your sample appear? If the CT difference is 10, don´t worry about the NTC as whatever stray signal that you may have will be less than 1 000th of your signal...and for sure you´ll not care about accuracy down to the third decimal if you are looking for fold change differences that are at least 1.5x... So if you do the math, even 6 cycle difference will not matter much.

Of course if it´s a question of no expression vs some expression you´d need  a completely blank NTC. However, that would not be the ideal negative control either, for that you´d need a -RT control to judge any stray signals that would come from gDNA et al. I´d recommend -RT control over NTC in any event, as it´s more powerful as a negative control; As doing -RT for 30-80 samples per experiment would be a bit costly, I usually pick 1-3 samples randomly to generate -RT controls and from their CTs (we use intron spanning primers, still sometimes one gets amplification from Qiagen RNease PLUS or TRIZOL purified RNA) I have at least an estimate where the "background" is approximately.

Jose Maria Saugar @ I do melt curve analysis for every reaction. NTC Tm is almost near to Tm of ample amplification. The difference is in threshold value which is more than 30 for NTC.

Serdar Tuncer @ thanks for your suggestions

Christoph Metzendorf @ thanks for you suggestions. Yes, I am using SYBR green methodology and usually get Ct around 20-25 of my samples and 31-35 for NTC.

Madhu Beta @ thanks. We use only nuclease free water. Our machine is calibrated properly. Even, we check background signalling for each well.

Hi, how you prepare you PCR reactions is well described except one thing - do you work in a PCR box (or yet better - one box for reaction, one for template)? We experienced a contamination coming from the air in the lab.

Jan Sterba @ thanks. we will try to follow your suggestion. 

Wassim Eid @ thanks for your reply. I went through this and got an exact same band in NTC. Although, band intensity is weak in NTC.

Madhu Beta@ we tried in all kinds of ways. We have changed SYBR green by same company as well as other company but problem is still persisting. Anyway after getting many responses, we decided to change our primers and see the results.

Why not run the experiment in another Real TIme PCR machine different from the one you are currently using. Just try it!

Dear Vikram,

My question is the melting peak of amplification u are getting at 31-35 same as your PCR product..? If No ; then its the behavior of primers you can increase annealing temperature.

You can check the primer efficiency with template dilutions if its near 80-100% you are good (R ^2 val=0.98 -1), but then make sure annealing Tm of dimers is different.

If Tm is same as Template change primers.

Thanks Mam. We have tried with dilutions and different annealing. The problem was primer -dimer.