Posted 01 June 2009 - 12:06 PM
Posted 01 June 2009 - 02:28 PM
We are doing RT-PCR after plasmid tranfection, RNA extraction and DNasa treatment. The problem is that we are not able to eliminate all the plasmid and we have amplification using only RNA. So I think We could digest with a restriction enzyme cutting into the gen before RT-PCR to avoid false positive amplification. What do you think??
Posted 02 June 2009 - 08:01 AM
Posted 02 June 2009 - 01:50 PM
DNAse would be the normal choice. With your strategy you would need to worry about the RE staying around in your RT reaction, and cutting the final dsDNA product after amplification. If you can heat kill the RE, then this might be an OK strategy. I'd try the DNAse first.
Actually, what we are doing is trying with DNasa, then digesting with the RE, and then heat inactivation before RT.
The problem, as I told you is that using the DNasa treatment is not enough, as we canít eliminate all the plasmid. We tried two different enzymes and double digestion time with no success.