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Serial Dilution Problens


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#1 Raff03

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Posted 23 February 2014 - 08:03 AM

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.   



#2 Epigeneticist

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Posted 25 February 2014 - 07:37 AM

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? 


Edited by Epigeneticist, 25 February 2014 - 07:41 AM.





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