A problem of quantitative PCR - (Aug/21/2005 )
I isolated mRNA from cells and did RT-PCR, there's a major band with some weak bands after running the gel, and i guess the primer design is not perfect to observe only one band. since i cannot get single band even though the intensity of the major one is much higher than the others, i am wondering if i can use this primer to perform quantitative PCR, thx,
Short answer: No
For quantitative PCR, you need a single band so that you can ensure that all the change in fluorescence is due to your product, not any non-specific product. Have you tried optimising your PCR using Mg2+ and/or temperature gradients to see if you can get a single band?
Can different Mg2+ concentrations improve band quality by eliminating those non-specific bands?? because before i only tried to use different Mg2+ concentrations to get bands when there is no band after running the gel.