Standard curve - Heeeeeelp (Oct/25/2005 )
I´m trying to construct a standard curve, I want to now if is necessary to introduce a fragment of a control gene in a plasmid vector, and how can I calculate the copy number of the plasmid?. What others alternatives exist to construct the standard curve?, and which one is the best if I want to quantify genes with very low level of expression in a single cell.
recently, it was posted that qRT PCR was feasible in one single cell...
it was molonco and it's here
for determining of copy number, hybridation on chromosome is a hard technique but works for that purpose...
To determine copy number, figure out the number of moles of dna in ther tube per ul (this is related to the length of the DNA as well as the concentration. There are links to the equations on this website. Then multiply by avagadro's number to figure out the number of molecules per ul. This will give you your copy number per ul of DNA. To make the standard curve, always store your DNA at high concentration and avoid freeze thaw cycles I have found. Make serial dilutions fresh each time you run a reaction.
I think it is best to introduce the gen in a control vector for this purpose. For one-step RT-PCR, I use an in vitro transcribed template.