These are likely so-called "primer-dimers" (some unspecific products amplified from primers only).

I am not sure if you are doing qPCR. But if so, then the amplification of such unspecific products in no problem as long as their Ct values are 2-3 cycles larger than the ct referring to your lower limit of quantification (say your least concentrated "positive" sample gives a Ct of 27 and the Ct of your NTCs is at about 30 or higher, that everything is fine).

If the Cts are too low (like your positives have Cts around 31, and at this cycle also the NTCs come up), then you have a problem that need to be solved. You could try to use a higher annealing temperature, lower primer concentration, lower Mg++ concentration. If this does not help, you need different primers. Alternatively there might be the possibility to use 10-50 times the amount of cDNA (this would derease the Ct of your sample, givin again the separation to the NTCs) - but this approach can be difficult (not enough sample available, inhibitory effect or other undesired side-effects due to the high concentration of cDNA buffer/salts or other incedients carried over into the PCR.

If your aim is a sensitive detection of rare copies (not quantification!) then you can set a temperature for measuring the signal above the melting temp. of the unspecific products. The signal of the resulting amplification curves will be lower and somewaht more noisy, but there won't be any exponential increas visible in reactions only amplifying unspecific products (on the other hand, a melting curve analysis can also do the job, so such tricks are often unneccesary).

Dear Jochen Wilhelm,

Thank you so much for your prompt response!

I will check the ct values to know if could be the reason why blank control is amplifying.

Jochen Wilhelm gave you the comprehensive details which will help you; however, i will recommend you to Increase your annealing temperature (1-2 degree). Usually we can avoid the primer dimer at high temperature. I personally found this helpful. 

Can you give some more details of the experiment? Are these multiplex reactions or individual tubes?. Also explain what is "blank" - what it contains.

Thank you Imran Ullah!

Manoj, I prepared one tube for each duplicate samples. Blanks are negative control. These samples contain only mix (b Act and specific markers), i.e. Reverse and Foward primers (0,33 ul each), SYBR green (10ul) and sterile H20 Mq (7,54ul) totalizing 20 ul per well. What do you think?

So, I guess it is a multiplex PCR, because above numbers do not add to 20 (0.33+0.33 + 7.54 +10). And if it is more than duplex (more than 2 pairs of primers in same tube), a lot of primer dimers can happen, as Jochen described.

If it is not a very well designed assay, you can have better success by doing each one in separate tube.

Yes, if this is a multiplex assay, there are many different primers (all in rather high concentrations) and a lot of mess can happen. If modestly elevating the annealing temperature does not help then you may need other primers - or, if you can do without multiplexing, try to make separate singleplex PCRs instead, as M N suggested.

Manoj,

I didn't add different primers in a same tube. I didnt use multiple primer sets within a single PCR mixture. I mean, for each primer pair I put in a same tube (MIX), for example, VEGF (F and R primers, H2O and SyBr Green) in a tube, AT1 in other tube, etc. After this, I mixed this with my samples to analyse. Negative control was only R and F primers, H2O and SyBr Green. I used the same plate to put these different  Mixes but each mix in different well. Intriguingly, only the negative control has primer dimer. The samples amplify very well.  Besides, it is happening  now but before it was ok.

It is not uncommon that only the negative controls show an accumulation of priomer-dimers. In these reactions there is no template to be amplified, all resources (primers and enzyme) remain unused all the time. When some unfortunate priming and elongation event happens, than this gets amplified.

In positive samples you have rather many template molecules from the very start that will be amplified with certainty. Thsi amplification will easily compete out an amplification of primer-dimers.

(this is gradual; samples with very low template concentrations can show a parallel  amplification of both, specific target and primer-dimers)

So you may see the occurence of primer-dimers as a "sign of boredom" of the polymerase...

Agree with Jochen. If some dimers are seen in NTC, but all your samples amplify well even at low copy inputs - you can best ignore it.

At what Ct do you see NTC signal? Is this near to your samples? If yes, you need to optimize further or go for a probe based system (eg: taqman). Remember that SYBR green may increase dimer due to increasing effective Tm. Intercalating dyes does that. Try increasing the annealing temp up by 4-5 degC, to compensate for this and use only hot-start enzymes.

I essentially agree with M N except for the that you may "go for a probe based system" (I assume he meant sequence-specific probes like hydrolysis or hybridization probes, mol. beacons or scorpoin primers etc;  because SBYR Green is also a probe - just not a sequence-specific one).

If you have an amplification of unspecific products that interfere with the amplification of your specific product, then this will impact the amplification efficiency and so invalidate the Ct value (and the entire quantification). Ignoring this by only looking at the signal of a sequence-specific probe will not change this - so you get wrong results and you even do not recognize it. Sequence-specific probes have advantages for multiplexing and for detection, but not for quantification.

But as Clynton described he presumably has not the problem that the amplification of primer-dimers interfere with the amplification of specific products. So he does not need to think about all this.

Again, shortly:

There is no problem when the negative controls amplify something that is not the specific product, and when the samples amplify nothing else but the specific product.

Thank you all for useful input!!!!!