I had to quantify the amount of bacterial DNA in some environmental samples that included genomic DNA from all kingdoms. I had no available pure E. coli or any other pure species DNA.
I used some DNA from one of the environmental samples that I was supposed to quantify the bacterial DNA in, and used it as a template for full length 16S gene amplification in a normal PCR reaction. After amplification, the amount of amplicon was measured with fluorescence nano drop as accurately as possible. This amplicon of now-known concentration was used to prepare the standard dilutions for the qPCR standard curve and to produce the curves for all the environmental samples. The qPCR was performed with a set of general 16S qPCR primer pair of ~150bp.
The question is, is this way of preparing a standard for this experiment correct and why might it be wrong? Would copy number affect the results in any way considering that both the full leng 16S and the qPCR 16S fragments are amplified with general bacterial primers? Is there any scientific papers published using such a method for bacterial DNA quantification.
yes your procedure is right infact this is the same procedure we use to find GM contamination in foods, or residual DNA analysis. Standard in qPCR by definition means sample whose quantity is matched with fluorescence et known quantity and known fluorescence. Refer SA bustin PCR revolution. Two practical inputs
1. Try to PCR purify (PCR clean up or gel extraction) the amplicon and then do qubit reading for quantity.
2. since u r amplifying the amplicons of standards so make standard of 10 to 12 points of diluition (refer my paper snapshot of kutch metagenome)
Sandeep, thank you so much for you answer and your advice. If you have any published papers to suggest that use this technique, could you please send me titles and authors?
Thank you!
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