Getting Rid of DNA after Transcription - (Aug/18/2011 )
I am working with a pool of oligos. I first amplify them then transcribe into RNA. I then use this RNA in a co-ip to recover RNA bound to a specific protein. The problem is, I am not able to get rid of the original starting material (amplified DNA) that was not transcribed. RT-PCR of the enriched RNA from the CO-IP shows multiple band product (see image). I think that this may be due to amplification of contaminating DNA that I tried to get rid of after transcription.
I have used DNase treatment after transcription with varying conditions to try to optimize the enzyme. Dnase does not work very well. I have also tried spermidine to get rid of DNA and it did not work at all for me. Some people us LiCl.
Does anyone have a tested protocol that works really well to get rid of DNA?
DNase generally doesn't get rid of all the DNA. Turbo DNase from Ambion may work better than standard DNase (I haven't tried it). I have heard that DNA-free (also from Ambion) is the best way to remove DNA from RNA samples.