still gDNA after DNase treatment??:( - (Aug/11/2005 )

Hi,

I isolated RNA from chicken brain.Although the kit (Roche High pure RNA isolation kit)contains DNase treatment step, it didn't work.So I did another DNase treatment( promega RQ 1 DNase I ) after RNA isolation but there are still some gDNA.So I repeat treatment once more, but I couldn't get rid of it I did PCR by using RNA as template and on agarose gel I saw smear ( gDNA ) on it.I attach gel photos.

09.08.05 --->on the right side of marker / after dnase treatment
08.08.05 --->second well / before dnase treatment

What should I do?? Please help me!!! Thank you...

Did you add Mg? Do you know that your DNase is active? Try doing another control where you use another DNA source (say your DNA marker). Do two parallel DNAase treatments, one on marker DNA only and one on your RNA sample with marker DNA added.

Daniel

DNA sequencing programs

Hi Daniel,

DNase buffer contains Mg at 1,5 mM conct but I didn't add extra Mg.

I will try these 2 parallel DNase reaction.Thank you for your answer:)


Aysegul Yildiz

Hi,

I use DNase stop solution that is activated by heating at 65 C after DNase treatment of my purified RNA but there are still some gDNA .So I repeated the treatment one more time.Most of gDNA are eliminated but there are still some.I think it can be caused by the stop solution added at the previous DNase treatment.May be it inhibits enzyme activity.If it is possible, is there any way to remove stop solution before the second treatment?

Thank you for your help!!!

This is likely the problem. I suggest EtOH precipitating your sample to clean it up.

Daniel

Save BigDye mix

Hi Daniel,

Thank you for the answer I will try it and tell you result...

Hi Daniel,

I isolated new RNA, it looks very very good on gel and there is no gDNA !!!:). The kit contains DNase itself but there, it is said RNA should be incubated with DNase for 15 min.@ RT.I think it was not enough so I waited for 45 min. and it is ok!

I also attach the gel photo.

Thank you

Hi Katanin

Yes your RNA looks very good. You may have had more DNA than normal in your initial prep or the DNase may be partially inactive. Remember even if you can't see any DNA you may still have enough traces left to interfere with RT-PCR.

Daniel

Longer, quality DNA sequencing

Hi Daniel,

Yes you are right that there may be still some gDNA.So I did a PCR using RNA as template to see if there is any product from gDNA and I couldn't see anything:)I think it seems ok for now...I want to attach this photo too but I forgot to take a copy form computer

Thank you and see you:)