Two bands on a denaturing gel - always two bands on a denaturing gel with double stranded substrate? (Oct/23/2005 )
i am using an enzyme assay in which a 21 mer p32 labeled substrate is cleaved at a single site. the substrate is double-stranded, and incubated with the enzyme for 30 min at 37. when i run the samples on a 20% denaturing gel, in the control (DNA only) lane, i see two bands. In the sample well (DNA with enzyme) i see the same two bands, with another: the lower (smaller) band has been cleaved, yielding a third band just underneath. now i'm guessing the second band is the top strand which contains the cleavage site (the enzyme makes single strand cuts). The distance between the first and second bands is large, both in the DNA only and sample lanes. This makes sense in the sample wells since if the top strand is being cleaved, it will be much smaller (about 8 mer) than the bottom and hence run smaller. However, i cannot explain the huge distance between the bands in the DNA only lane. I know this is a denaturing gel, but other gels in publications i have seen with similar substrates and the same enzyme only have one band in the DNA only lane, and two in the sample lanes: substrate and cleaved product. The top strand of the substrate contains an abasic site (uracil to abasic by UDG) and i'm wondering if it is somehow breaking. i'd appreciate any input as to what these two bands in the DNA only lane are... and if they are just the top and bottom strands of the DNA (denaturing gel) ...then how come other denaturing gels in publications show only one band with a similar double-stranded substrate? thanks a bunch!
I'm just curious.
-What is the origin of your DNA substrate?
-What is the aim of having a denaturing gel?