problem with optimized protocol - (May/25/2007 )

hi everyone
i have optimized a PCR for a gene it gave really good results with different samples but now i m facing a big problem that when i used do the PCR reaction with the same conditions i get the smear instead of a proper band of DNA, i have already changed my Primer dilution twice, buffer, Taq polymerase enzyme, dNTPs, water but i m getting the same results even i have tried to re-amplify the PCR which i get positive there there was a clear band but again there is only smear.
i m very worried about it because i have to submit a report to my supervisor as soon as possible, can any one help me in this regard
saba

Try diluting your template 10x and 100x.

I assume that your DNA template is genomic DNA.

another thing you should do is run some of your uncut template DNA out on a 1% agarose gel. This is to check that your DNA is still in good shape and have not yet degraded.l

If the DNA is good, it should form a high molecular weight band with little or no downward streaking. The more downward streaking, the more low molecular weight DNA is present, and thus the more degraded the DNA is.