I am currently trying to do a school project trying to dilute a 50% V600E mutation down to 0%, using pyrosequencing to determine the percentage mutation. The aim was to start off with a 1:2 dilution series of the V600E cell line: WT DNA. However, when I am doing this,the first sample which is the cell line is showing a 50% mutation, then the rest of the samples are just 100% wildtype. I double checked that I had added in the mutated DNA and checked that the concentration of the WT and the V600E cell line is the same before I started the dilution. I was just wondering if anyone could offer any other advice as to what could be causing this?
My method has been to made 100ul stock solution of WT DNA at a concentration of 15ng/ul (same as V600E cell line). I have nanodropped the stock solution after to make sure it is the right concentration.
I then did 10ul WT DNA:10ul V600E Cell line to get a 25% mutation, then 10ul of this solution with 10ul WT DNA to get a 12.5% dilution etc...
I always give the solution a gentle mix with the pipette before making up the next dilution.
I just can't understand why when I make up the 25% mutation it is coming up as 100% WT on the pyrosequencing run. The answer is probably staring me in the face but I just cannot see it! Any help would be much appreciated.