Hi there,
I am working on airborne microbes. I want to know the concentration of bacteria in my sample before I go for NGS. Since it is an environmental sample it might have a lot of mixed bacterial species. I am planning to do qPCR for overall bacteria and for positive and negative separately by using a specific primer and probe. I am stuck at standard curve preparation now as I need to prepare three standard curves. Since my sample is air and it might have a lot of species, I don't know how to proceed now. Can anyone help me with this?
For the standard curve it can be good to take a known concentration of DNA for the primer set. For example purify DNA from one bacterial strain that the primer will work on if possible. Then you will want to do serial dilutions. I would suggest doing a 1/3, 1/10, 1/30, 1/100, 1/300. Ideally if you are able to start with a known concentration then you can determine exactly how much DNA you have in each sample.
Hi Joshua thanks for your answer. Initially, I was planning for that but later got one confusion that is, my sample has mixed DNA, will it opt to compare it with a standard curve prepared from the single DNA? Joshua Beytebiere
The standard is one sequence, you quantified it and you use it to make a standard curve for this sequence. For each different sequence you need an own separate standard.
Your sample is mixture of different sequences, and to quantify one particular sequence you use the primers for that particular sequence. This can be compared to the standard for that sequence. The same way you can quantify all the other sequences in the sample, each in comparison to its own standard.
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