Im doing gel electrophoresis to detect if the mutant gene is expressed in my experimtal mice. if the desired mutant gene is not present, i donot see any bnd. if present i see. but there is a difference in the band intensities.
Hi Madhavi,
The first, and easiest, thing that comes to mind is to make sure you are using the same amount of template DNA in each reaction. Quantify your template DNA, dilute to ~50ng/uL and use 1uL per reaction.
Hi Madhavi,
if everything is calibrated properly but you are doing PCR bellow the saturative cycle number (i.e. less than 30 cycles), the band intensity could reflect a difference in gene expression level (sometimes we do like that, and we call it "semi-quantitative RT-PCR). If your mutant is null mutant, you expect no band on the gel, if you use heterozygotes as control, they might have reduced amount of mRNA than a wild-type control. This is one explanation for the band intensity differences. But note, this works only if every other conditions (including the amount of template- what Barry suggested) are the same.
It can be due to :- 1. less amount of template DNA OR
2. Primer annealing temperature not sufficient
3. extension time not enough
If still there is this difference:-yes same gene with diff intensity shows differences in the amplification, the reason is the specificity. you can Quantify the DNA before start also make sure of the above factors.
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