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260/230 ratio, does it matter?


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#1 Wek

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Posted 26 April 2015 - 07:45 PM

After doing a gel purification with a Qiagen kit my insert/plasmid has a very low 260/230 ratio (<1) but a great 260/280 ratio. I have been told to ignore it and continue with my cloning. Does this happen to anyone else? I know for a fact it's not ethanol and most likely the salts from the solutions.


Edited by Wek, 26 April 2015 - 07:46 PM.


#2 r.rosati

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Posted 27 April 2015 - 03:24 AM

It does matter; how much depends on the application. I'd say that ligation and cloning might be affected, yes (but if you want to try, then I'd be curious to know if it worked...)

Why do you say it can't be ethanol? You probably have this low reading because the column didn't dry properly after the washing steps; and the wash buffer contains both salts and ethanol.



#3 Wek

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Posted 27 April 2015 - 05:59 AM

I don't think it is ethanol because I use multiple washes with each solution (qt, ph, and pe). I also use a manifold vacuum and during the last wash with pe I let the vacuum running for a little while to get excess ethanol sucked off, then spin in a microfuge at 14000g for 2 mins, and then I let it sit at RT for a min or two before eluting. I have no problem with plasmid preps.

Also I was told long ago that when purifying low nucleic acids concentrations you can get a low 260/230 ratio due to salts eluting and not much can be done about it which makes some sense. Does anyone what's the lowest concentration of nucleus acid I should purify when working with columns? I am trying to digest 1ug.

Thanks

#4 Trof

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Posted 28 April 2015 - 10:43 AM

It's the salts, and it happens with Q kits. Also true about the low concentrations, the are always "some" salts (in phenol purification "some phenol"), and the less NA you have, the worse the ratio.

 

Sometime increasing the final spin step in spin protocol to 3 minutes is as an optimization for getting as much ethanol out as possible, but you already did an excess of this.

 

But what kind of kit you use? I don't recognise the buffers.. PE is the common ethanol was buffer, for gel extractions you use either QIAQuick/MinElute or QIAEX and they do not have QT or PH buffers (it's QG for column kits). What do you use?
And there is just one important note about salts in the column protocol, after adding PE you should let it stand for 5 minutes before spin/vacuum.

 

And about your digest, you should be limited by the volume first. Depending in what volume you want to digest, you need to keep the concentration high enough to fit that volume. Since the highest volume you can get from QIAquick column is ~45 ul, then you can't have concentration below 22 ng and that is still horrible volume.

Sensitivity of RE to salts differ, some are quite sensitive, some not at all (they already use high-salt buffer). Bu amount of salts also increases with the ratio of DNA/reaction volume, logically, so you just want to have as high concentration as possible (i.e. using MinElute columns if your vector is below 4kb or what is in the manual..).


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#5 r.rosati

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Posted 29 April 2015 - 09:46 AM

I stand corrected, and agree that salts are the culprit in this case. I went for sources to back my knowledge and found out it was incorrect. "Devil" 's in the little things.

Thank you both!

Roberto






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