mchlbrmn wrote: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.