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Old 01-29-2015, 03:15 PM   #1
leftisthominid
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Default Ineffective Zymo Clean and Concentrate-5 kit

Hello, I have used a Zymo Clean and Concentrate-5 sample kit on three of my DNA samples extracted from FTA cards. Each three samples started with 12 ul of DNA suspended in reduced EDTA 1 TE (10mM Tris Ultrapure, pH 8.0, and 0.1mM EDTA.), which was purchased from Affymetrix.

The Zymo kit appears to have failed horribly, despite the fact that I have followed the protocol to the letter (though I did use the TE cited above instead of the provided elution buffer due to the pH difference). I was wondering if you had any suggestions regarding what is causing this. I have attached nanodrop data for the samples before and after Zymo clean-up. The "cleaned" samples have Zymo appended to their names and have markedly lower DNA concentrations and 260/230 ratios. Any one else have similar experiences?


Sample ID User ID Date Time ng/ul A260 A280 260/280 260/230
S038 Default 1/26/2015 6:28 PM 47.63 0.953 0.668 1.43 1.21
S038 Default 1/26/2015 6:29 PM 47.69 0.954 0.661 1.44 1.22
S038Zymo Default 1/29/2015 4:13 PM 21.5 0.43 0.269 1.6 0.46
S038Zymo Default 1/29/2015 4:14 PM 12.21 0.244 0.13 1.87 0.4

S043 Default 1/26/2015 6:38 PM 31.26 0.625 0.438 1.43 1.2
S043 Default 1/26/2015 6:39 PM 30.36 0.607 0.435 1.39 1.33
S043Zymo Default 1/29/2015 4:15 PM 5.73 0.115 0.043 2.68 0.7
S043Zymo Default 1/29/2015 4:15 PM 6.01 0.12 0.057 2.11 0.63

S054 Default 1/28/2015 6:21 PM 60.75 1.215 0.894 1.36 1.28
S054 Default 1/28/2015 6:22 PM 59.53 1.191 0.797 1.49 1.09
S054Zymo Default 1/29/2015 4:17 PM 18.9 0.378 0.195 1.94 0.94
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Old 01-29-2015, 11:45 PM   #2
nucacidhunter
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In my experience these columns do not perform as some other brands and seems to be leaky. I only use them for particular applications where removing strongly bound proteins to DNA is more important than yield. I guess that observed lower yield is partly due to the fact that Nanodrop quantifies nucleotides either in single format or as oligo in DNA and RNA. During purification most of the nucleotides, small DNA fragments, single stranded DNA and possibly RNA is lost which would decrease the Nanodrop reading considerably. The reason for low 260/230 is presence of salts from binding buffer. Washing with extra volume of wash buffer and also pipetting out flow through rather than decanting increases that ratio.
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Old 01-30-2015, 02:04 PM   #3
jhalpin
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Quote:
In my experience these columns do not perform as some other brands and seems to be leaky.
Is there a brand you'd recommend for DNA cleanup for sequencing applications?

Thanks,

Jessica
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Old 01-30-2015, 03:34 PM   #4
nucacidhunter
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I have not compare many brands, so I cannot recommend any particular brand. If you are looking for input genomic DNA clean up (it is important only if the first step in library prep is an enzymatic reaction), I have had good results with Zymo's gDNA columns. To increase recovery, one can elute with warm buffer at above 55C and incubate for 5 min at the same temperature. A second elution also increases yield by 10-15%. If you are following a protocol which shears DNA and then includes a clean up before end repair, cleaning input DNA does not make any difference to results.
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Old 02-02-2015, 06:04 AM   #5
pmiguel
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Just to reiterate -- UV spec is questionable method for determining the concentration of a DNA sample, especially genomic DNA. Far too many substances absorb at 260nm, confounding attempts to determine the concentration of long, double-stranded DNA in a solution. Worst of all, as nucacidhunter writes, are mono- and oligo-nucleotides and RNA--all absorb pretty much at the same place as long duplex DNA. And RNA amounts in a cell tend to vastly outstrip that of DNA.
I have compiled a list of some of the common lab chemical UV spectra here.

That said, another issue we see with Zymo DNA purification columns is that people sometimes attempt to use them on DNA samples that are not fully solvated. If the viscosity of your sample is fairly high, then the sample will not enter the matrix of the column. It will just sit on top. Of course the yield is then terrible.

Although it isn't really Zymo's fault that people do this, it happens enough that I think they should specifically address it in their instructions. I spent my first decade at the bench doing hundreds of genomic DNA preps. But some people really don't deal with long DNA much and don't think about the effect of viscosity on a column.

Finally, I really don't like adding hot buffer to genomic DNA. It is a common recommendation in kit instructions, but if you are making a library down-stream I think it will lead to a bias against AT-rich parts of the genome due to localized strand denaturation. Even for protocols where DNA is isolated from agarose by dissolving it with chaotropes, we find we get much better results just adding enough chaotrope to dissolve the agarose at room temperature rather than cranking the temps up.

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Old 02-02-2015, 11:40 PM   #6
nucacidhunter
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Quote:
Originally Posted by pmiguel View Post
Finally, I really don't like adding hot buffer to genomic DNA. It is a common recommendation in kit instructions, but if you are making a library down-stream I think it will lead to a bias against AT-rich parts of the genome due to localized strand denaturation. Even for protocols where DNA is isolated from agarose by dissolving it with chaotropes, we find we get much better results just adding enough chaotrope to dissolve the agarose at room temperature rather than cranking the temps up.

--
Phillip

Local denaturation of AT rich region due to warm elution buffer is less of a concern because genomic DNA fragments are large and those local region would be renatured when it cools down. Most gDNA extraction methods also involves incubation with extraction buffer in high temperatures, so gDNA surviving those temperatures are less likely to be affected with warm elution buffer. I think it is more of concern when small DNA fragments are purified from gel like early Illumina days where 150 bp fragments had to be purified from gel. Of course this an opinion and I have not seen any experimental evidence.
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Old 02-03-2015, 02:49 AM   #7
lorendarith
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I've used their kits successfully for Illumina library prep cleanups (regular and gel extraction), both performed ok or the same as the QIAGEN ones; maybe even a little better because there's no liquid left in the tube due to their design.

I've also used the genomic DNA kit (regular and gel extraction), this also worked alright with the prewarmed buffer.

If your yield from these columns is low, you can always try to elute several times from the same column and see if helps. You can also reapply the eluate to the column again in case there's a problem with the elution.

Why not try to use bead purification instead if columns are not working for you?
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Old 02-03-2015, 04:33 AM   #8
pmiguel
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Quote:
Originally Posted by nucacidhunter View Post
Local denaturation of AT rich region due to warm elution buffer is less of a concern because genomic DNA fragments are large and those local region would be renatured when it cools down. Most gDNA extraction methods also involves incubation with extraction buffer in high temperatures, so gDNA surviving those temperatures are less likely to be affected with warm elution buffer. I think it is more of concern when small DNA fragments are purified from gel like early Illumina days where 150 bp fragments had to be purified from gel. Of course this an opinion and I have not seen any experimental evidence.
Sure, good points.
To be honest, we just see better yields adding more chaotrope at room temp for agarose gel slice extractions. That could be for many reasons.

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