1 reply to this topic

[pcr_newbie]

Hello,
  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?

Thank you.

Attached Files

•  double bands.bmp   129.54K   78 downloads

[David C H]

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.