Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

standard curve using genomic dna - (Sep/09/2013 )



I've been working on generating a standard curve using genomic DNA with primers that target a promoter. I've done it a couple times but my slopes are very bad (~1) and R2 values ranging from 60 to 80. I know that the DNA stickiness (high concentration) may have an effect but I was wondering if the fact that the primers I'm using results in 1 large peak and 1 smaller peak has any influence on the standard curve.


Any insight on if this has any effect.




I'd suggest cutting up your genomic DNA before trying to do a serial dilution. Depending on your species, a rare cutter such as NotI may work. Otherwise, high pressure expression through a narrow needle several times will break the long strands in a random way, as will nebulizers. Uncut genomic DNA is essentially impossible to accurately dilute.