How to improve DNase efficiency?

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How to improve DNase efficiency?

Post by soam » Wed Jul 23, 2008 12:15 pm

Hi everyone,

I'm in a training period, in a laboratory of virology.

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

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