I have DNA extracted from air filters and wish to use qPCR to estimate the bacterial load. I have read through some papers but am stuck with some methodology.
I know I need to make a standard curve of known concentrations of DNA and how many cells there were and compare my values Ct values from my unknowns to get the # of cells but I do not know how to go about doing that.
Thank you for any help!
At first step you have to determine your bacteria that you have on filter.(culture or NGS, depends on goals of your study)
Then make a standard curve.
It is really better to choose a gene that has one copy number.
Also when you choose this protocol you can't differentiate between live and dead cells.
Firstly, you need to determine the presence of your targeted bacterial cell/genome using culture or PCR or NGS as said by Alireza.
Secondly, you can make a standard curve for multiplex PCR using gBlock gene fragments. I am attaching here the link for tips to develop standard curve using gBlock:
https://sg.idtdna.com/pages/education/decoded/article/tips-for-working-with-gblocks-gene-fragments
- Badges
- Science topic
- Similar topics
- Papers