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
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.