I have isolated cDNA and it look ok after PCR using housekeeping genes. Anyhow as soon as I try primers (blasted etc) for the gene of intrest there is no or a wrong product... even primer that used to work for genomic PCR seem not to work at all anymore.
So anyhow I was wondering what to change, I tried optimizing with temperature or Mg or Q Solution but still nothing. I read reverse transcriptase inhibits Taq. So do you people clean up your product from reverse transcription or use it directly for pcr amplification? Do I need to change PCR conditions if using cDNA instead of genomic DNA?
Slowly this starts to become really frustrating. So I would be really THANKFULL if anyone here had any ideas how to improve this...