High 260/230 ratio for DNA

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Kristina Mlinac...
Kristina Mlinac Jerkovic's picture
High 260/230 ratio for DNA

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?

Shampa's picture
 You are right it should be

 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.

Jitendra Sinha
Jitendra Sinha's picture
I may be wrong also. Shouldn

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's picture
I would advice diluting your

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. 

Monu's picture
 I also suspect the high

 I also suspect the high salts conc is the main reason behind it. But you should check spec instrument functioning also.

LBurger's picture
 Wouldn't a very high 260/230

 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 Mlinac...
Kristina Mlinac Jerkovic's picture
After concentrating my

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
Susie Q's picture
I`m working with AFLPs, so I

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's picture
As others have already said,

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's picture
So, I have a low 260/230 (0

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 Delgado
Ivan Delgado's picture
A low 260/230 ratio can be

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.

arodri03's picture
So I can go ahead with my

So I can go ahead with my pGEM protocol then? Or try eluting it in warm water instead of buffer?

Ivan Delgado
Ivan Delgado's picture
You can go ahead with any

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.

Rajeshwari patel
Rajeshwari patel's picture
hi arodri03,

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's picture
I am ethanol precipitating it

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

Ivan Delgado
Ivan Delgado's picture
I agree that performing an

I agree that performing an ethanol precipitation is the way to go. If you do that take this into consideration: you may lose a fair amount of your DNA when you ethanol precipitate. Hopefully you will get enough DNA back. If you don't, and your downstream experiment does not work, simply perform a new DNA extraction and this time elute with warm water.

Good luck

Carogar's picture
 Hi Kristina,

 Hi Kristina,
Would you please tell me your protocol for RNA concentration?
Thank you!