Strange signals in the NTC - (Jun/14/2014 )

Dear All

I still getting signals in the NTC ( CT around 25 ) even at varous primer concentration  while my target is around (24, 25). the TM of my primer is 59 so i used different annealing 55, 57 Is is really strange 

 I did the following to avoid getting it but still no luck : 

1- Changes place of work 

2. change tips, pippets 

3. Dilute the primer again 

4. use other SYBR green tube 

5. tried various annealing temp 

6. reduce cycle number 

I think this is not primer dimer as the CT is high and this is not contamination. What this might be ? 

If this primer dimer, how to avoid it 

You can check the melt curve analysis of NTC and compare it with your target. Also compare the Tm value of your NTC peak in Melt curve to ensure that what you get is not primer dimers.