I want to compare the Traditional CFU count of bacteria with Real-time PCR method, but I m having some issues with the making standard curve. So please if anyone is willing to help I will really appreciate it.
Hi Ibrahim,
At the very beginning, I would like to clarify that, there are no such methods available till now which can use DNA as a standard for the absolute quantification of RNA, because there are no such controls are yet known which can check the efficiency of the reverse transcription. But yes if you want to correlate your CFU count with RT-PCR, probably you can use some housekeeping gene as a control. I guess the below-mentioned link can help you for your better understanding.
Kind regards
Debasish
http://www6.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf
Hi
I just can say that choosing a gene that bacteria had one copy number of that and it is not related to RNA absolutely.
your subject have a big question mark, I think, such as when you use a gene for counting you not able make difference between live and dead bacteria.
Consider all aspects.
good luck.
Traditionally, you choose a gene that is single copy in the genome and clone it into a plasmid. You do a plasmid DNA prep, calculate the concentration of DNA in your extraction, and use serial dilutions to create your standard curve. You will need to know the size of your plasmid backbone and the size of your inserted gene of interest. Good luck!
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