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Nanodrop question for RNA


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12 replies to this topic

#1 molbio1234

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Posted 10 June 2014 - 12:00 PM

I have got a reading from RNA extracted sample with the following

A260/280  = 2.08

A260/230 = 2.26

A260 = 14.924

A230 = 7.191

 

Do these readings show good RNA?

 

 



#2 molbio1234

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Posted 11 June 2014 - 06:05 AM

does anyone know? I believe readings are good but some websites say 260/280 = 1.8-2, and 260/230 of 2.0, so I wanted to get other opinions on RNA.



#3 labbey2.0

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Posted 12 June 2014 - 07:43 AM

I think your absorbance values are fine.  From my experience, having an OD260/OD280 of 2.0 is clean RNA; I would be hesitant to use anything less than 1.8.  If you're in doubt, I would suggest running a denaturing gel and check what you see there.



#4 Tabaluga

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Posted 12 June 2014 - 08:51 AM

Looks fine to me as well. The 280/260 is roughly within range, and as for the 260/230 I'd rather be worried about too low values anyway (contamination).


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#5 bob1

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Posted 12 June 2014 - 01:04 PM

The absorbance readings depend on the solvent - if you used water, then 1.9-2.0 is the accepted range for 260:280, but values differ for different buffered systems.



#6 Bio-Lad

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Posted 16 June 2014 - 06:02 PM

The absorbance readings depend on the solvent - if you used water, then 1.9-2.0 is the accepted range for 260:280, but values differ for different buffered systems.

 

Bob1, could you elaborate a bit on this?  I'm definitely no nanodrop expert (I use TE or water only) and thought that blanking with the buffer/solvent would eliminate much of the differences.  What accounts for the difference in ranges?  Thanks!


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#7 bob1

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Posted 17 June 2014 - 01:16 AM

I can't remember exactly but I think something to do with protonation of the purines and pyramidines of the DNA.



#8 Trof

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Posted 17 June 2014 - 02:23 AM

I just remembered this TechNote.

http://www.lifetechn...tating-rna.html


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#9 Truz89

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Posted 10 July 2014 - 07:51 AM

I have a similar, I have no experience with nanodrop, but I have extracted DNA using the QIAGEN kits and the 260/280 results range from 2.6-3.3. And the average DNA concentration is 122946 ug/ul 

I'm not sure what to make of those numbers, since they are higher than usual.


Edited by Truz89, 10 July 2014 - 07:53 AM.


#10 bob1

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Posted 10 July 2014 - 01:02 PM

I have a similar, I have no experience with nanodrop, but I have extracted DNA using the QIAGEN kits and the 260/280 results range from 2.6-3.3. And the average DNA concentration is 122946 ug/ul 

I'm not sure what to make of those numbers, since they are higher than usual.

Check the DNA on a gel - results like that usually indicate contaminants of some sort.

 

The curve shown by the nanodrop should give you some idea if the samples are clean.

 

Incidentally, the nanodrop measures in ng/ul not ug/ul...



#11 Truz89

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Posted 10 July 2014 - 03:33 PM

 

I have a similar, I have no experience with nanodrop, but I have extracted DNA using the QIAGEN kits and the 260/280 results range from 2.6-3.3. And the average DNA concentration is 122946 ug/ul 

I'm not sure what to make of those numbers, since they are higher than usual.

Check the DNA on a gel - results like that usually indicate contaminants of some sort.

 

The curve shown by the nanodrop should give you some idea if the samples are clean.

 

Incidentally, the nanodrop measures in ng/ul not ug/ul...

 

Just to make sure, the DNA extracted is from urine. Would that make a difference or does the 1.8 - 2 "rule" still apply?

 

thanks

 



#12 bob1

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Posted 10 July 2014 - 04:54 PM

It still applies - the ratio is dependent on the solvent of the DNA, not the source of the DNA.  Urine has lots of salts that might be relatively hard to remove, these could easily affect the extraction of the DNA and what you can do with it once you have extracted it.



#13 Truz89

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Posted 11 July 2014 - 07:06 AM

It still applies - the ratio is dependent on the solvent of the DNA, not the source of the DNA.  Urine has lots of salts that might be relatively hard to remove, these could easily affect the extraction of the DNA and what you can do with it once you have extracted it.

 

ok thank you!






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