cDNA PCR - no product - questions - HELP!!!!

[Stjarna]

Hello,

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...

[danfive]

Originally Posted by Stjarna

I read reverse transcriptase inhibits Taq. So do you people clean up your product from reverse transcription or use it directly for pcr amplification?

Never heard of this before, I don't believe it---never had it cause any problems. No clean up step needed.

Originally Posted by Stjarna

Do I need to change PCR conditions if using cDNA instead of genomic DNA?

Generally no, unless you want to change the primer concentration. How do you know that the primers are in transcribed region? Or that the gene is actively transcribed in this cell type?