It is likely that either your DNA template and/or PCR primers have degraded since your initial successful PCR reaction. Resuspend primer stocks in TE buffer, aliquote and store at -20. Dilute working primer concentrations from the primer stocks in nuclease free water. Store diluted stocks at 4 degrees for 1 to 2 weeks. Avoid excessive freeze-thaw cycles of DNA template (store at-20 long term) and primer stocks. Your best bet is to get new primers made and resuspend and store as above.

Good luck

dear sean 

I checked my primers and other materials , all of them are ok. 

I hvae nonspecific bands with my specific band and my problem is how to remove them .

oh yes i did before , I ve done alot to remove nonspecific bands but they are staying here but getting weak . I need to remove them completely.

im looking for new idea can help me .

but I did it before and im sure my components are not contaminated.

thanks every body for helping me .Im still looking for new idea that can help me 

Did you tried to increase the temperature in the cycle? Normally lower temperatures than the "ideal ones" lead to the increase of non-specific bands. If you increase the temperature it could resolve the problem.

I hope it helps

You can try  DMSO to remove non-specific bands.

thanks every body

using a new aliquot of DNA is a good idea and also try putting up gradient pcr again. a gel pic and your PCR protocol will be helpful here for people to troubleshoot further. 

2-8% of DMSO in your PCR reaction can eliminate your non specific bands or else I suggest you to cut the band of your interest and reamplify using that as your template. It should work fine....

If you are sure that none of your reagents are contaminated, using fresh, RNAse-treated DNA might be a good idea. Removing unwanted parts from a PCR gel can be not soo reliable. PCR are too susceptible to contaminators. Aim perfection and start with new DNA. 

Although I don`t know all details of your PCR assay, I assume you have no contaminations in your reagents. Of course, it depends on your target, but which NA contamination will produce several unspecific fragments of various sizes with the same PCR primer set?

As Nuno mentioned, I assume the PCR conditions are not stringent enough. Try to i) increase the annealing temperature, ii) shorten elongation time, iii) reduce primer concentration, iv) reduce number of PCR cycles.

Vary only one parameter per iteration. The kinetic of the specific product will be favoured over the misprimed amplification artifacts.

One further advice: Double-check the PCR protocol of your cycler. Maybe your lab mate accidently changed some parameters in the thermoprotocol.

I think that surly you have to change your instructions, but you should be very careful about your sterility conditions, make your tubes in the ice when you're preparing you master mix, be sure that you don't spend a lot of time when preparing your solutions, be sure that your melting temperature is good and suitable, check the primers percentage of CG, the length of your primers? hope you find the mistake or give us more details to can help you.

thanks all

finally i forced to extract my bind from gel!!! cuase nothing else worked