QIAquick Absorption Peak at 230nm rather than 260

[skblazer]

Hello,

Each time, I used QIAquick gel extraction kit to extract the size selection product, the OD 260/230 is extremely weird (very low, close to 0), and 260/280 is a little bit higher (2.0-2.1). I found the absorption peak was moved from 260nm to 230nm, but the DNA should be extracted. I guess the remaining salt (such as EDTA) causes this, but I've tried to optimize the gel extraction step for several times (such as adding 6X volumes of QG, extending incubating time for QG, standing 5min after adding PE, ...), but they didn't work.

The colleagues at lab also have this problem, but it didn't affect their TOPO cloning or Sanger sequencing.

I have no idea if such weird ratio will affect some steps in the Illumina paired-end library preparation, such as Phusion PCR reaction or Cluster generation?

Anyone has such experience?

Many thanks,
SK

[ECO]

It's the guanidium-SCN in the QG. An extra (or even a third) PE wash can help. In my experience it can affect efficiencies of sensitive enzymatic reactions, but is not a huge problem for amplification based preps.

[pmiguel]

If it is a peak at 230 nm, that probably is a guanidium salt. But if it is somewhat shorter it may be acetate (eg, sodium acetate). At high concentrations absorbs there. (I can dig up the reference and post it here, if anyone want me to...)

The difference is probably crucial. Sodium acetate is just a salt, but guanidium salts are powerful denaturants.

--
Phillip

[skblazer]

I' sorry, it's the peak shorter, it's just significantly higher at 230 than 260. (please see attached)

So the sodium acetate should not be a problem, am I right?

Originally Posted by pmiguel

If it is a peak at 230 nm, that probably is a guanidium salt. But if it is somewhat shorter it may be acetate (eg, sodium acetate). At high concentrations absorbs there. (I can dig up the reference and post it here, if anyone want me to...)

The difference is probably crucial. Sodium acetate is just a salt, but guanidium salts are powerful denaturants.

--
Phillip

[skblazer]

I'm sorry I described inappropriately, it's not a peak at 230, it's just 260/230 is very low.

Originally Posted by ECO

It's the guanidium-SCN in the QG. An extra (or even a third) PE wash can help. In my experience it can affect efficiencies of sensitive enzymatic reactions, but is not a huge problem for amplification based preps.

[Cofactor Genomics]

In my experience, this type of curve is not unusual to see with the gel extraction kit. The moderator is correct, you should introduce more washes with PE than the manual states. This will help mitigate this peak, however it probably will not get rid of it completely. Normally, I would usually follow-up with some kind of buffer exchange (Ampure beads, Microcon, or drop dialysis), but I was always concerned about losing more sample and potentially introducing more bias. Thus, I would usually go with drop dialysis. Not sure many people do that anymore or are comfortable floating their sample on a dialysis disk in a bin of water.

[pmiguel]

Originally Posted by skblazer

I' sorry, it's the peak shorter, it's just significantly higher at 230 than 260. (please see attached)

So the sodium acetate should not be a problem, am I right?

That looks like sodium acetate to me. A good paper on the UV spectrum of aqueous acetate is this one:

Reference Type: Journal Article
Author: Ruderman, Graciela
Author: Caffarena, Ernesto R.
Author: Mogilner, Inés G.
Author: Tolosa, Eduardo J.
Primary Title: Hydrogen Bonding of Carboxylic Acids in Aqueous Solutions—UV Spectroscopy, Viscosity, and Molecular Simulation of Acetic Acid
Journal Name: Journal of Solution Chemistry
Cover Date: 1998-10-21
Publisher: Springer Netherlands
Issn: 0095-9782
Subject: Chemistry and Materials Science
Start Page: 935
End Page: 948
Volume: 27
Issue: 10
Url: http://dx.doi.org/10.1023/A:1022615329598
Doi: 10.1023/A:1022615329598

Is it a problem? Well to be able to see acetate on a nanodrop you probably need to be at 100 mM or more. It is not as bad as having guanidium salts in your sample for most downstream applications. But it can definitely cause problems to have 100 mM salt in your sample.

--
Phillip

[skblazer]

Thanks for the advice. I'll try washing twice by PE.

Originally Posted by Cofactor Genomics

In my experience, this type of curve is not unusual to see with the gel extraction kit. The moderator is correct, you should introduce more washes with PE than the manual states. This will help mitigate this peak, however it probably will not get rid of it completely. Normally, I would usually follow-up with some kind of buffer exchange (Ampure beads, Microcon, or drop dialysis), but I was always concerned about losing more sample and potentially introducing more bias. Thus, I would usually go with drop dialysis. Not sure many people do that anymore or are comfortable floating their sample on a dialysis disk in a bin of water.

[skblazer]

Hi Phillip,

Many thanks for the paper.

But do you know where the huge sodium acetate come from? Is it from buffer QG to buffer the PH? But I didn't hear a lot of people report this. I'll try wash the columns twice by PE.

Originally Posted by pmiguel

That looks like sodium acetate to me. A good paper on the UV spectrum of aqueous acetate is this one:

Reference Type: Journal Article
Author: Ruderman, Graciela
Author: Caffarena, Ernesto R.
Author: Mogilner, Inés G.
Author: Tolosa, Eduardo J.
Primary Title: Hydrogen Bonding of Carboxylic Acids in Aqueous Solutions—UV Spectroscopy, Viscosity, and Molecular Simulation of Acetic Acid
Journal Name: Journal of Solution Chemistry
Cover Date: 1998-10-21
Publisher: Springer Netherlands
Issn: 0095-9782
Subject: Chemistry and Materials Science
Start Page: 935
End Page: 948
Volume: 27
Issue: 10
Url: http://dx.doi.org/10.1023/A:1022615329598
Doi: 10.1023/A:1022615329598

Is it a problem? Well to be able to see acetate on a nanodrop you probably need to be at 100 mM or more. It is not as bad as having guanidium salts in your sample for most downstream applications. But it can definitely cause problems to have 100 mM salt in your sample.

--
Phillip

[aureias]

Hi, I recently just saw what you have seen.

The first image is from a nanodrop: Comparison of what a 1 wash PE looks like compared to straight qiaquick purification w/o gel elution.

2nd image: parallel amplicon, except this time 5x wash through a vacuum manifold. I really think its the Buffer QG that is effecting this. I'm not sure how to completely get rid of it, but the multiple washes does seem to remove it somewhat. I'm going to try to sequence it and see if the quality is sufficient.

Please let me know if you've found a way of purifying it out completely. Thanks!

[pmiguel]

If it is really bothering you, just dilute the sample to ~500ul and concentrate it with a microcon spin column. Re-dilute a few times (on the same column), if necessary. That will get rid of acetate and/or guanidium.

But if you are getting a large peak like that, there is probably a technique issue. You might want to call Qiagen and ask for advice on how to avoid the carry over.

--
Phillip

[pmiguel]

Originally Posted by aureias

Hi, I recently just saw what you have seen.

The first image is from a nanodrop: Comparison of what a 1 wash PE looks like compared to straight qiaquick purification w/o gel elution.

This is what a 5x wash through a vacuum manifold looks like of a gel fragment. I really think its the Buffer QG that is effecting this. I'm not sure how to completely get rid of it, but the multiple washes does seem to remove it somewhat. I'm going to try to sequence it and see if the quality is sufficient.

Wait, I just re-read this. What are samples on the two runs? On the first picture I see a green peak centered around 230 nm, and a black peak around 260 nm. Which is which?

--
Phillip

[aureias]

ahh sorry.

first image is a comparison of PCR amplicon purifications with either a pcr purification qiaquick or else a pcr gel purification qiaquick using standard recommended protocol.

Qiaquick Gel Purification (green line) (buffer QG)
other lines: Qiaquick PCR purification without gel extraction (buffer PB)

second image was a parallel gel purification with 5x washes. It cleared up some of the peak, but still not as clean as I'd like.

[aureias]

Hmm, gave them a call. As expected, its the salts from QG.

A few things they said might improve it is:

longer incubations in the wash buffer
incubations at 37 degrees in the wash buffer
multiple washes

I've done multiple washes, and that seems to eliminate it a fair bit, not completely, but somewhat.

I'm going to give the other options a try and will see what pops out.

[pmiguel]

When we see that 230 nm peak (guanidine isothiocyanate), we just do a microcon. But we use those spin filters moderately frequently, so they are available.

--
Phillip

[mjj16]

Reviving a slightly old thread but this same problem happened to me (unfortunately before I read this). Is there a way I can further clean my samples that are already gel purified via Qiaquick? Can I apply the eluted samples back on a Qiaquick column and perform the extra washes (with incubation) as suggested here? Or does anyone have another other suggestions on how to remove the salts post Qiaquick?

Thanks in advance for any help!

[Bilgenur]

Hi all,

Very productive discussion indeed. I am experiencing a similar problem. I did DNA extraction from water samples using phenol/chloroform. However, I do get a shoulder/high peak at 230 nm whereas there is a slight peak at 260 nm. My 230:260:280 ratio is more like this: 1.6:0.8:0.5.

I adapted my fieldwork sampling protocol from Foote et al. (2012)*. Based on the literature search, and similar problems people have experienced, I do think that the high peak at 230 nm is due to some salt contamination along with residual ethanol. I am in the midst of troubleshooting, but I am not sure how to fix this problem. There are people suggesting to use ether (to remove organic solvents) or to dialyse the probe if salt is the contaminant. Also, during PCR some additional reagents can be used, such as BSA, glycerol to prevent any potential inhibitors getting amplified.

Any suggestions on how to carry out? I would like to have purified DNA before troubleshooting with PCR step, ideally.

*http://www.plosone.org/article/info%...l.pone.0041781

[nucacidhunter]

Using 1.8x Ampure or similar beads are good options and amenable to high throughput methods. If you have high molecular weight DNA, you can elute with warm buffer with longer incubation to minimise loss.

[pmiguel]

Originally Posted by nucacidhunter

Using 1.8x Ampure or similar beads are good options and amenable to high throughput methods. If you have high molecular weight DNA, you can elute with warm buffer with longer incubation to minimise loss.

Agree.
Look, the problem here isn't that the poster doesn't have a desirable A230:A260 ratio. The problem is that the poster's prep methodology left contaminating salts mixed in with their "purified" sample. Some of these salts have a UV spectrum that you see when you look. Chances are that if you had assays that could detect non-optically active substances, you would see lots of stuff there as well.
For example see this post.
Does it matter? Who knows. Generally a bunch of optically active substances showing up in your sample isn't a desired outcome, but there are simply too many factors involved to say.
So my tendency is to say stop using UV spec. Use a Qubit instead.

--
Phillip

[JBKri]

I am facing a very similar problem to Bilgenur. We are doing DNA extraction from water samples using phenol/chloroform, and we get a (very) high peak at ~230 nm. We also have sometimes a smaller peak at ~270 which is probably phenol. The pellet we see after ethanol precipitation is large and greyish to white, and I suspect it is mostly salts rather than DNA.

Originally Posted by nucacidhunter

Using 1.8x Ampure or similar beads are good options and amenable to high throughput methods. If you have high molecular weight DNA, you can elute with warm buffer with longer incubation to minimise loss.

I did try a 1.8x AMpure cleanup, but for some reason we lost all the DNA. I speculate that the contaminants in the extracted DNA (phenol, salts,..?) interfered with the properties of the AMpure buffer and prevented the binding of DNA to the beads. Or did I overdry the beads (~2 min)?