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What is the protocol to remove RNA contamination (Using RNase) from DNA?


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#1 Huongnguyen.miss

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Posted 03 June 2014 - 06:12 AM

What should be the concentration of RNase to remove RNA from DNA? And after removal of RNA how to denature RNase from DNA sample?

 

Any help will be apprecipated, thanks so much in advance :)



#2 bob1

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Posted 03 June 2014 - 01:11 PM

This is usually done after the lysis of the cells to get at the DNA - make your lysate, salt out most of the proteins, add RNase, incubate 30 min at 37 C.  Then do a phenol-chloroform extraction or two, and proceed from there,



#3 Huongnguyen.miss

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Posted 03 June 2014 - 06:53 PM

i did same like you said above, but when i checked by Agarose gel and spectrophotometry are still same previous =((



#4 bob1

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Posted 03 June 2014 - 09:19 PM

Neither of those methods will tell you if it is RNA definitively.  RNA and DNA differ only in the absence of oxygen groups in the DNA that are present in RNA so they look more or less identical to a spectrophotometer.  On gel electrophoresis, degraded DNA looks just like RNA - the EtBr intercalates in the same manner to both - can you post a gel picture so we can see what is going on?

 

It may be that you have so much RNA present that you can't get rid of it all, you could try adding more RNase and re-extracting.



#5 Huongnguyen.miss

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Posted 04 June 2014 - 11:25 PM

this pic is my result . Lane 1 and 2 from left to right hand is my Bacterial DNA. ( others are not).

do you have any ideas about that? 

Attached Thumbnails

  • IMG_1100.JPG


#6 phage434

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Posted 05 June 2014 - 06:23 AM

Looks like genomic DNA to me. There may be some RNA, but why do you care? I don't think there is a lot.



#7 Huongnguyen.miss

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Posted 07 June 2014 - 04:31 AM

'coz i need that to sent for sequencing to get whole genome of bacteria. so company required me get more purity :(



#8 phage434

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Posted 07 June 2014 - 07:29 AM

RNA is essentially ignored in genomic DNA sequencing preparation protocols. Its major effect will be in misleading quantification if you are using spectrometer readings. Did your sequencing center complain about this sample?



#9 Huongnguyen.miss

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Posted 07 June 2014 - 11:00 PM

i have not sent yet, but my professor who has not satisfied about this result :(






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