I am preparing DNA standard curve using gag cloned plasmid by SYBR green assay. I have optimized my primers concentration (250nM) and prepared different DNA concentration based on the copy number. I am getting 10 fold difference till 104 dilutions but after that the difference is minimal (i.e 1.6, 2.0 Ct value difference). May I please know what all conditions should I focus on to get better standard curve and difference of 3 Ct value to get an efficiency of 90-110% and slope =-3.2.
It seems you are losing the DNA of lower dilutions. Are you using any carrier DNA or RNA for diluting your plasmid? If not it is highly advisable. DNA is known to bind to plastic surfaces which affect the concentration.
Thanks for your reply. I have not used any carrier DNA for the standard. I will try with calf thymus DNA. Also, I could not observe any primer dimer in the background. Melting curve is showing perfect peak till 104 dilutions.After 104 dilutions my Ct value is not showing fold difference. I mean the values are almost same(Ct = 22,23) but not to undetectable level (high Ct value).
- Badges
- Science topic