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Serial Dilution Problens - (Feb/23/2014 )

Hi All, 


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.   


Does your cell line have a heterozygous or homozygous V600E mutation? Sounds like it is heterozygous. Have you done pyrosequencing on the wild type and V600E samples alone to verify them?