Estimated abundance of cells in extracted DNA

[esbena]

Hi everyone.

I´m pretty new to this qPCR method and could really use some help.

I have performed qPCR on DNA (16S rRNA genes + amoA genes ) extracted from environmental sample (biofilm) with both bacteria and archaea primers.

My problem is that I do not know how to estimate the abundance of bacterial and archaeal template copy numbers in the original sample.

I have constructed a standard curve from crossing point (Cp) data and copies/microliter template (log). But I simply cannot figure out how to do it.

So if someone could explain in detail (and I mean detail) which numbers I shall use and what to do, it would be perfect.

I have some info:

Average bacteria/Archaea harbors 4.07 and 1.76 16SrRNA gene copies/genome, respectively.

Average bacteria/Archaea harbors 2.5 and 1.0 amoA gene copies/genome, respectively.

My Cp values: Bacteria 16SrRNA: 17.15 (10x dilution), amoA: 24 (1x dilution)
Archaea 16SrRNA: 31.0 (10x dilution), amoA: 30 (1x dilution)
2 microliter template/reaction.

If there is any info I´ve forgotten please remind me.

Huge thanks.

/Troubled student, Aarhus University, Denmark.

[Mikael Kubista]

Test GenEx (www.multid.se). It estimates also copy numbers from standard curves including confidence intervals!