problems with gDNA doing real time PCR in yeast - (Sep/17/2013 )
I`d like to perform real time PCR experiments in yeast. The problem is contamining genomic DNA within the isolated RNA which leads to artificial amplification products after cDNA synthesis. DNAse digestion didnt solve the problem properly and since most yeast genes dont have introns i am limited in primer design to overcome this issue.
Did anybody here make comparable experiences and has any idea how to get rid of the DNA completely or to avoid amplification from the DNA?
How do you know DNAse digestion didn't work? How do you do it?
If I use the DNAse treated RNA (prior to cDNA synthesis) as template in a standard PCR with gene specific primers i get "perfect" products...that is my standard control for DNA contamination.