I am developing a method to measure the number of cells in a sample from an anaerobic culture using qPCR, and I want to use gDNA as a standard. I am wondering if anybody has any advice about how to do this. I have been doing serial dilutions of both cells and gDNA, and I am getting okay results. I think I can calculate the amount of DNA per cell, figure out how many cells are in each well, figure out the 16S copy number, and compare it to the DNA. I am using e coli right now. I appreciate any advice about this.
Have you found that your gDNA curve and your cell-derived DNA curves have different slopes? If so, there is a mathematical way to reconcile the 2 universes and calculate copy number of the cell gDNA. Hopefully the operon number is the same for 16S in both the gDNA standard and the e coli? Or, are you also analyzing an assumed "single-copy" gene here somewhere?
If you need to 'reconcile the 2 universes' mentioned above - the attached has worked in these situations.
Dear Elizabeth....I wonder if you managed to set up your methodology . I am actually facing the same problem in assessing the number of bacterial cells by qPCR. I appreciate if you feed me back with your experience.
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