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Repurifying RNA after extraction - No Kits

RNA purification nanodrop repurification

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5 replies to this topic

#1 Mad Researcher

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Posted 19 May 2015 - 05:36 AM

Hello,

 

I extracted RNA from brain samples today. I checked for the purity on the nanodrop but the 260/280 ratio is 1.79. I want to know if there is any method (apart from commercially available kits) to repurify my RNA again. Please let me know of any protocol that can be used to purify it again (if available)

 

PS: stupid question - Will repurifying my RNA again will increase the yield of my RNA?

 

Thanks.


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Mad Researcher

#2 bob1

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Posted 19 May 2015 - 12:58 PM

What's wrong with a 1.79 reading? Which solution is your RNA in... this determines the correct ratio for pure RNA

 

Re-purifying RNA will result in a loss of somewhere between 20 and 80% usually. You can use a standard phenol-chloroform extraction (trizol and the like) to do this.



#3 Mad Researcher

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Posted 22 May 2015 - 01:37 PM

What's wrong with a 1.79 reading? Which solution is your RNA in... this determines the correct ratio for pure RNA

 

Re-purifying RNA will result in a loss of somewhere between 20 and 80% usually. You can use a standard phenol-chloroform extraction (trizol and the like) to do this.

Sorry for my late response. Shouldn't the purity of the RNA be between 2.0-2.2 (nanodrop reading)?


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Mad Researcher

#4 bob1

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Posted 22 May 2015 - 06:24 PM

Possibly... it depends on the solution it is in. Those ratios work for water IIRC, but not other solutions.



#5 Mad Researcher

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Posted 23 May 2015 - 12:58 AM

The RNA is in water. What do you suggest should the RNA be eluted in TE or water?


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#6 bob1

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Posted 25 May 2015 - 12:51 PM

TE would be more stable than water for RNA. Whether the ratio is important or not depends on what you intend to do with the sample. The only difference between the RNA and DNA calulations is DNA is multiplied by 40, while RNA is multiplied by 50... they are the same calculation otherwise, so a low ratio does not necessarily indicate DNA contamination of your RNA.

 

Also note that the original publications for these ratios are for checking RNA and DNA contamination of protein solutions, not the other way around. The ratio thing works well for contamination of protein solutions, but not necessarily well for protein contamination of RNA/DNA.







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