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Problem standard curve. qPCr

3C assay. Ligation

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#1 Lilip

Lilip

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Posted 06 November 2013 - 03:07 AM

I have been trying to perform the control template for 3C assay which consists of amplifying from the gDNA small fragments ( your controls) purify from the gel, put them together in equimolar concentration, digest and  ligate (to get all the possible combinations).
 
After that I'm  supposed to performe a standard curve using this DNA template to check the primer efficiency.
 
The thing is: for the standard curve the first dilution has a low ct ( around 20) and the second ( 10x) goes to 32, the third goes to around 34 and the forth and fifth do not amplify. I repeated many times and got the same pattern. I started with more diluted dna, but then I get amplification just in the first cycles and sometimes the second dilution gives me a lower CT value than the first.
 
The only thing i did different from my collegue ( that has it working) was the ligation step. I did in a larger volume ( of course adjusting all the reagents), but the DNA was in a lower concentration compared to my colegue. Does it make a difference? Does it explain this weird result from the aplification for standard curve?
 
Thanks a lot.





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