smearing below band of interest

[jamestoon1]

i am doing touchdown pcr with the following condition:

98'C 30 sec initial denaturation

10 cycles:
98'C 5 sec denaturation
60'C 5 sec annealing
72'C 5 sec extension

25 cycles:
98'C 5 sec denaturation
59'C 5 sec annealing
72'C 5 sec extension

72'C 2 min final extension

my band of interest is 200bp
i got the band
but there are smearing below the band:
[IMG=http://img688.imageshack.us/img688/3035/85488077.jpg][/IMG]

what has gone wrong and how should i rectify the problem?

FYI, my pcr master mix contain 18.5ul master mix, 0.5 ul forward primer, 0.5 ul reverse primer, 0.5 ul DNA.

[mchlbrmn]

I don't know the kind of gel you used, so is it possible the small molecular weight is just primers? If you run an uncycled lane you would see.
Otherwise, the normal thing to increase specificity is to increase the anneal temperature. In my experience one or two degrees often accomplishes little, unless you are lucky and are right aroung the actual (not the predicted) anneal temperature. If you have a gradient machine you could try several temperatures at once.
I notice the well is bright. If you used more template than normal that could contribute to a smear also.

[jamestoon1]

I don't know the kind of gel you used, so is it possible the small molecular weight is just primers? If you run an uncycled lane you would see.
Otherwise, the normal thing to increase specificity is to increase the anneal temperature. In my experience one or two degrees often accomplishes little, unless you are lucky and are right aroung the actual (not the predicted) anneal temperature. If you have a gradient machine you could try several temperatures at once.
I notice the well is bright. If you used more template than normal that could contribute to a smear also.

I was using 2% agarose gel.
I have run a gradient pcr, 60'C appears to be the highest I can go. Above 60'C, the band will disappear altogether.
And in the present touchdown pcr, i chose 59'C as the lower temp because from my optimization experiments, 59'C can result in multiple bands, so i would not go below that temp.
And the template is not more than normal. I used only 0.5ul. The DNA conc is approx 50 ng/ul, which means I used 25ng/ul as starting material. Moreover, in my other PCR reactions (using different primer pairs), even 1.0ul or sometimes 2.0ul template works fine.
There should be no problem with primer design. i have BLASTed and have checked with a few softwares. Moreover this primer pair is obtained from literature. A lot of people have used this primer pair.

[snettisham]

This streaking my be due to poor DNA quality. I have seen this before. My guess is that the streaking could be simply the original template DNA that has been chopped up by vortexing, or restriction enzyme contamination, or maybe star activity of Proteinase K. Thus, the streaking is not an amplified product.
To show this, try running a single DNA sample with and without the primers over a DNA gradient, maybe .25, .5, 1, and 2 ul, and see if there is a gradient effect of DNA on the smear vs band intensity. Also, if you can, run DNA from a different source that has cleanly worked on a different PCR AND always run a no DNA control. If you conclude that the smear is due to the DNA quality perhaps reevaluate your DNA preparation or can you live with the smear? Good luck.