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High 260/230 ratio for DNA

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Kristina M.
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Topic Started by Kristina M.
on 7/3/2009 1:22 AM   
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I purified my DNA samples form whole blood using QIAamp Blood Maxi Kit (Spin Protocol). Using Nanodrop, my 260/280 ratio was ok (around 1.8), but 260/230 ratio in some samples was as high as 8! I know that ideal 260/230 ratio is around 2, and if it is lower that probably means contamination with organic solvents. Does anybody  knows what  high ratio means?


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Shampa
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Posted By Shampa
on 7/3/2009 2:58 AM   
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 You are right it should be aroung 1.5 ideally. What I know from my previous experience is that the high ratio indicates high salt concentration in your sample. Please try to reduce it.



Jiten
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Posted By Jiten
on 7/3/2009 16:41 PM   
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I may be wrong also. Shouldn't we expect the A260 / A230 ratio to be low if the salt concentration is high in the samples? I have experienced this problem and I have not been able to sort out what the error happening with similar A260 / A230 ratios. I have got even once at 18.



sps
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Posted By sps
on 7/3/2009 3:30 AM   
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I would advice diluting your sample in 10mM Tris-HCl at pH 7.4 but not water. One more thing, low ratios may be due to phenol contamination. Check it out!!! High ratios might come from salts in yoour samples. I never saw ratios as high as indicated here. But if you used Trizol, maybe try doing clean up using a column spin methods and try again. Wish you good luck. 

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Monu
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Posted By Monu
on 7/3/2009 3:54 AM   
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 I also suspect the high salts conc is the main reason behind it. But you should check spec instrument functioning also.

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LBurger
United States

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Posted By LBurger
on 11/9/2009 10:18 AM   
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 Wouldn't a very high 260/230 ratio indicate a low (in in the case of some samples I just OD'd zero) absorbance at A230. Personally, I'm more concerned with contamination issues when I have significant absorbance at A230 indicating phenol, salt, etc (and low 260/230) than when it's too high. From what I've read anything over 2 is good.

Low A230, could also be related to how dilute your sample is; if the A260 is low (say 0.05), then it may be difficult to accurately estimate A230 resulting in over inflated 260/230 ratios. 

I'd remeasure the samples more concentrated, if possible, and see if you can get a better A230 reading. I like to shoot for an A260 of ~0.1.  However, as I said above I think a higher ratio (low A230) is better than low.



Kristina M.
Croatia

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Posted By Kristina M.
on 11/12/2009 2:56 AM   
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After concentrating my samples (using ethanol precipitation after adition of 3M sodium acetate) and re-measuring the concentration, my A260/230 ratio was normal! I suppose the reason for the high ratio could be high initial salt concentration, but whatever it was it is gone after this procedure



Susie Q
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Posted By Susie Q
on 12/13/2009 16:56 PM   
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I`m working with AFLPs, so I need a nice DNA that could be completely digested. I have seen that DNAs with low 260/230 ratios don`t have fully digestion... it is logical... but also DNAs with High 260/230 ratios (more than 4) just parcially digest. I am wondering myself why this could be happening????



asdquet
Canada

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Posted By asdquet
on 1/12/2011 9:04 AM   
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As others have already said, the high ratios are not due to salt contamination.  Salt contamination causes LOW ratios.  Since the ratios become normal after concentrating the DNA, it's likely that it was too dilute to be read properly by your spec and therefore caused non-sensical ratios.



arodri03
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Posted By arodri03
on 7/5/2011 12:45 PM   
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So, I have a low 260/230 (0.03) but good 260/280 (2.97)
I ran a PCR, cut out the band and extracted using the QIAgen gel extraction kit. Why is the 260/280 okay
but the 260/230 not so okay? High salt? Sould I try an ethanol precipitation protocol?



Ivan
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Posted By Ivan
on 7/5/2011 13:51 PM   
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A low 260/230 ratio can be due to a lot of reasons. Even EDTA, which is present in TE buffer, absorbs at 230 nm. My guess is that whatever was used to elute your DNA, typically TE, is the reason why your 260/230 is this low.

I would not worry about it.


Ivan Delgado Orlic Carlsbad, CA



arodri03
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Posted By arodri03
on 7/5/2011 15:41 PM   
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So I can go ahead with my pGEM protocol then? Or try eluting it in warm water instead of buffer?



Ivan
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Posted By Ivan
on 7/5/2011 16:40 PM   
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You can go ahead with any protocol, unless the protocol is inhibited by the presence of whatever is giving you a low 260/230 ratio. Unfortunately eluting your DNA using an elution buffer like TE, provided by many DNA purification kits, does not mean the DNA will be useful for downstream applications. You should take into consideration what your downstream applications will be before choosing what elution buffer to use.


Ivan Delgado Orlic Carlsbad, CA




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Posted By Rajeshwari patel
on 7/6/2011 3:30 AM   
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hi arodri03,

What ever IVAN is saying it is true. But  in this condition I would go for the ethanol precipitation and try to elute in warm water.



arodri03
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Posted By arodri03
on 7/6/2011 7:39 AM   
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I am ethanol precipitating it now and then will elute in warm water. Thanks for all the help- I'll let you know if it doesn't work. haha



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