I need some advice/opinions.
Basically I did PCR to amplify some beef DNA, and then ran a gel a few times. Now the problem is last time I didnt get no band at all, and this time I see a slight faint band, the size of it is correct but it is hardly there and looks like a primer dimer.
The reagents i am using for PCR are: Promega Master mix
which includes 50 units/ml of Taq DNA polymerase supplied in a proprietary reaction buffer (pH 8.5), 400μM
dATP, 400μM dGTP, 400μM dCTP, 400μM dTTP, 3mM MgCl2
The quantity I am using is 12.5 ul.
Then, Dh20 6.5 in positive tube, and 7.5 in negative tube
My forward and reverse primers at 2.5ul for both tubes
and then lastly DNA in positive tube at 1.0ul
and we also use the mineral oil (50ul) for the PCR to stop evaporation etc
After, this I load 15ul of my samples on the gel (agarose gel is 1% made up of 0.5 agarose and 50ml 1xTBE) alongside the ladder. I use 5ul of the loading dye for each sample.
The DNA ladder comes out fine each time I do it.
Where am I going wrong???