How to improve DNase efficiency? - (Jul/27/2008 )

Hi everyone,


Using in vitro transcription, we synthetized a ~8 kb RNA, and then we used DNase to get rid of all DNA. After that, we purified our RNAs. By PCR and electrophoresis, we found out that we still had DNA with our RNA.

Therefore we treated again with DNase and then purified, but it appears (by real-time PCR) that not all the DNA was digested.

We first used the RNase free DNase 1 (2U/µl), mixing 1µl with 19µl of the products of the in vitro transcription, and during 15 minutes at 37°C.
The second time, we mixed 1µl with ~99µl of "purified" RNA, still 15 minutes at 37°C.

What do you think could be done to improve the efficiency of the digestion step with the DNase?

Thank you

Before purifying my RNA I check it on a gel to ensure the DNAse has done its job. For some reason I find that doing the incubation at 37C in a water bath is not as efficeient as an incubator (I havent tested this this, just an experience thing). Also the DNAase I use says one unit will digest 1ug DNA in 15 min at 37C but I generally do mine for one hour.

You could check your DNAase by just digesting some DNA over a few time periods to see whats best.

Hope it works out