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gDNA still presents even after DNase treatment


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#1 Shahira

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Posted 03 April 2014 - 04:21 PM

Extracting RNA from Staphylococcus epidermidis using Qiagen RNeasy kit, then I DNase the samples using promega DNase and do qRT-pcr....but gDNA still present.

 

When i have tried qiagen DNase and Turbo DNase in different experments, gDNA still presents also.

 

I don't know what to do to get ride of gDNA....if staphylococcus DNA is so hard to get ride of????

 

Thanks in advance.

 

 


Edited by Shahira, 03 April 2014 - 04:26 PM.


#2 merlav

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Posted 04 April 2014 - 04:41 AM

Try to use the RNeasy plus, it have a column for DNA elimination, then do the Dnase step that should eliminate any trace of dna


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#3 Shahira

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Posted 04 April 2014 - 05:49 AM

Thanks for the reply

 

I already have RNeasy mini kit (qiagen), it contains columns for RNA extraction . Can i use this column to make DNA elimination on it or it requires another specific column?



#4 Trof

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Posted 04 April 2014 - 10:57 AM

Sorry it's not clear.. did you tried TURBO DNase after the column DNase treatment?

 

We found the Q Dnase to be a bit leaky on DNA eradication, but DnaseFree is also limited by the amount of DNA it can digest. If you have high starting concentration of DNA, using both DNases should eat more of than each one separately.

 

(BTW are you sure the bacterial DNA is definitely in your sample and not a comtamination from elsewhere?)


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#5 Shahira

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Posted 04 April 2014 - 01:27 PM

Thanks Trof

 

i didn't use column for DNA elimination before....i was using DNase ... i have tried 3 different enzymes separatly (Qiagen, promega  & turbo). all of them didn't eliminate DNA completely except for the promega . But when doing real time for second time using promega DNase, DNA returns back.



#6 merlav

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Posted 06 April 2014 - 05:00 PM

The plus kit have an extra column for DNA elimination. If you can't change the kit, can do the DNase step 2x or use a cDNA kit that contains a DNA elimination step prior the RT. Make sure that you are using material certified for RNA extraction, that those supplies are for use of RNA only, that you are cleaning area, pipettes, gloves (using RNA zap or RNA eliminase or similar) moments before the extraction. Use always filter tips and the box that is in use label it "RNA extraction only"

Edited by merlav, 06 April 2014 - 05:07 PM.

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#7 phage434

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Posted 06 April 2014 - 06:39 PM

You should run a negative control for your PCR reaction (one without any template). You could be seeing carryover DNA contamination of one of your reagents. This would make it look like you have DNA in your reactions.






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