Dnase digestion after Invitro transcription
c224 at gmx.de
Sun Nov 10 15:16:02 EST 2002
I did some invitro transcription reactions with E. coli RNA polymerase.
After invitro transcription I purify the RNA using Qiagen's RNeasy Kit
and then I use the RNA as a template in RT-PCR.
I had some problems with the digestion of my template DNA after in vitro
First I tried to add the Dnase immediately after the IVT and kept it at
37 °C for further 30 minutes. After that I purified the RNA.
The reaction volume was 15 µl. The digestion didn't work at all, I
examined that in a PCR without Reverse Transcriptase.
Could RNA polymerase (or anything else) inhibit DNAse? There's no EDTA
in the reaction. Is it necessary to inactivate RNA polymerase and how
would I do it?
Next time I purified the RNA after IVT. I added Dnase to the purified
RNA, kept it at 37°C for 30 minutes and heated it to 95°C for 5 minutes.
I took this as a template for RT-PCR. A control PCR showed that the
digestion was only partly successful.
In a control, I mixed DNA and Dnase and incubated at 37°C for 30
minutes. After heat denaturation of DNAse (5min, 95°C), the following
PCR yielded no product. So, the Dnase is fine.
Do you have any idea how to improve the DNase digestion?
Thanks in advance,
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