| |||||||||
| Login :: Register :: Search forums :: Top Users :: Reagent |
|
Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
|
Whether there is RNA contaminated in extracted genomic DNA?
Posted by: biomedlover (IP Hidden, Junior member, 11)
Date: July 18, 2005 04:16AM
Human genomic DNA was extracted from whole blood following the Protocol 6, Chapter 6 in 3rd edition of Molecular Cloning. There are great differences for the quantity of DNA extracted with or without RNase A treatment.
In my protocols, first one was fully followed above protocol, that means after the WBC pellets was digested with proteinase K, it was treated with DNase-free RNase A; Second one was exclude the treatment with RNase A. Properties of genomic DNA having been treated by RNase A: The A260/A280 ration was 1.84 ¨C 1.86, totally 3.75 ug DNA could be obtained from 300 ul whole blood. Properties of genomic DNA WITHOUT treatment by RNase A: The A260/A280 ration was 1.82 ¨C 1.84, totally 24 ug DNA could be obtained from 300 ul whole blood. The preparation of RNase A: RNase A was purchased from Roche. The 10 mg powder was dissolved in 500 ul TE Buffer (pH8.0) following 10 min boiling water bath to inactivated the possible contaminated DNase. And then the concentration of RNase was adjusted to 4.0 mg/ml, and 3.0 ul RNase A (4.0mg/ml) was used for every 300 ul whole blood to degrade the RNA. My questions is: 1. Why there are so great differences for the DNA quantity between above two protocols? 2. For genomic DNA without treated with RNA, is there any contaminated RNA? Of my known, the A260/A280 ration of RNA should be great than 1.9. In my results, the ration is always between 1.82-1.84, did this indicated that there should be no contamination of the RNA?
Re: Whether there is RNA contaminated in extracted genomic DNA?
Posted by: mito lab (IP Hidden, Unregistered user, )
Date: July 18, 2005 01:33PM
Did you run agarose gel electrophoresis for your genomic DNA? There is no way to distinguish DNA/RNA by A260/A280 since they are both nucleic acid. If genomic DNA isn't degraded by your home made DNase then you shouldn't see much smearing but the high molecular weight genomic DNA. Then I wouldn't worry too much about the yield. There will always be trace of RNA contamination depending on your protocol. A high quality DNase free RNase will help to eliminate it.
Re: Whether there is RNA contaminated in extracted genomic DNA?
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: July 19, 2005 07:20AM
you cannot tell whether a DNA sample is contaminated with RNA by A260/A280 as both absorb at the same wavelength. that being said i find it hard to believe that the difference between the quanitity of DNA extracted in the two samples (with and without RNase) is due to RNA contamination alone. the difference is simply too much. I think instead that for whatever reason you simply got a better yield with your second sample; some of this increase may be due to RNA contamination. i wouldnt worry about it either as mitolab suggests.
Re: Whether there is RNA contaminated in extracted genomic DNA?
Posted by: biomedlover (IP Hidden, Junior member, 11)
Date: July 19, 2005 08:12PM
Agarose gel electrophoresis was performed. There was no smear bands.
for additional, DNA that was extracted without treatment of RNase A was post-extraction digested with RNase A, and then the A260, A260/A280 ratio was detected. The results showed no decrease of both DNA conctration and A260/A280 ratio. did this indicate that there's no contamination of RNA?
Re: Whether there is RNA contaminated in extracted genomic DNA?
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: July 20, 2005 09:42AM
yes
Thanks for femmeauburn
Posted by: biomedlover (IP Hidden, Junior member, 11)
Date: July 20, 2005 09:51PM
Thanks a lot
Re: Thanks for femmeauburn
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: July 21, 2005 08:44AM
you're quite welcome. hope everything goes well.
|
We are moving ... Please post to our new community forums
|
|
||||||||