qPCR standards - (May/17/2006 )
Hi ! I am a beginner in qPCR and I am setting up my assay using Syber Green dye. I have no problem of dimers or aspecific peaks so I have done a serial dilution of a purified plasmid containig the target to construct the standard curve. The curve is good ma efficiency is a bit low (about 83%).
I would like to know:
- Have I to PCR my standards every time with the samples or once I have the standard curve can I use it as reference for quantification.
- I have not linearized the plasmid for the standard curve. Should I ?
- How can I improve my efficiency (using higher primer concentration ? Now is 600 nM in 25ul reaction).
Thanks for your replies !
Here is my answer
1. is better for you to PCR you standard every time with yor sample to eliminate the error between each run.
2. No need to linearized you plasmid.
3. Efficeincy of 85% is not good enough. Since your primer is o.6uM which i think is eough.... you might think of adding more MgCl2. You can perform a serial of MgCl2 concentratin from 1.5, 2.0 ..... 5.0mM with 0.5mM every interval. Chose the MgCl2 concentration which give you highest efficiency whitout giving you primer dimer and unspecific binding....