I am doing a quantification experiment for c.perfringens from fecal samples.I am following a paper which describes that DNA was isolated from the stool sample by Qiagen stool kit.After that they have done the real time PCR by taking 5ul of the isolated DNA and quantified against a standard curve. Now ,how to back calculate it? As whatever we will get it will be for 5ul of the DNA sample.In the kit protocol samples undergo dilution at different steps. Moreover I have also done CFU count both the results do not correspond to one another.
Comparison is well understood. But the question is if someone is taking 200mg of sample and ultimately get 200ul of DNA,is it the entire DNA representative of that 200mg?Because in the protocol the sample gets diluted many times.
Don't forget you will always lose 10-20% of your nucleic acid when you extract with a qiagen kit, this will also depend on your elution volume
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