As long as you have a band in the negative control you have to fight contamination. Use fresh solutions, use a different set of pipettes, ... Lanes 10 to 14 could be positive, but there is no proof. Sample 9 could be negative due to some inhibiting activity in your samples not present in the water control.

It's contamination or mistake

nope i did not mislabel them. Or I mislabeled them a hundred times, since it's not the only experiment i've done. My positive control just does not work anymore... It worked sometime but it is an old sample. 

Template DNA may inhibit PCR. Water-control containing no DNA therefore may give an unspecific Signal or amplify low contaminations. Try a run without any positive controls. If possible use diluted templates. Try higher annealing to avoid  (if this is the case) unspecific amplification. If it is an E.coli-System remember that Taq may contain residual E.coli DNA from Taq production.

I wish you success

I agree with Rene Koppel. I had experienced the similar frustration so here are few tips:  First try filter tips for everything starting from diluting your primer and to setup reaction because pipette is main source of contamination of DNA specifically when  others people are using the same sets of pipette. if you are not using filter tips so far then you have already contaminated all your PCR reagents and primers with your template so you should start over and use all the PCR reagents fresh and use filter tips.  

alright this is what i did:

- I put my eppendorf tubes, water, filter tips, pipettes in a PCR cabinet. Put the UV light on for 15 minutes. Used two different batches of primers. Diluted a new one just in case my old one was contaminated. Used new buffer, new dNTPs. However the same polymerase as i used before. 

samples:

mix of sample 9 with sample 12 to determine inhibiting substances in sample 9. 3 different mixes with new primers, old primers and nested primers. 

sample 12 which functions as my positive control since i know this sample works. again 3 different mixes with new primers old primers and nested primers

negative controls which contained water. mix with new primers old primers nested primers.

and a negative control which contained only the mastermix with new primers. 

however still the same result in my negative control. however this time the mix that contained the old primers i used in the experiment above shows no contamination. 

I am afraid that you have a problem with your primers.

We observed problems with primers (from literature too!). If used under unfavourable conditons primers can become a template themselves and make primer dimers and even larger products. Whether they are formed depends partly on the primer concentration, but it can be an inherent property of the primerpair. When your problem cannot be solved by primer titration (even unqual concentration of primers can be needed), you have to redesign your primers. We experienced this ourselves.

I presume you perform conventional PCR.

Then I agree with the suggestion of artur. However. shorter cylces can only be applied in balance to your input target in your positive samples. To inventorise his suggestion. Set 5 identical samples and take one out after e.g. 15, 20, 125, 30 and 35 cycles.

To inventorise the effect of primer concentration you have to do a checkerboard to bein with e.g. 30 pmol FW and reverse primers and then 60, 120, 240, 480 in every combination

Hello,

  After reading all that had been said by the others, I don't have much to add except that  I wanted to emphasize on contamination and quality of the product.

Best Regards,

Erick,

Are you sure you have seen the band  that you desire?

It is likely that Band in the gel can see It's not your band.