DNA loss on Bioanalyzer

moedog45 · 02-24-2012, 12:47 PM

About two weeks ago I sheared some gDNA samples on the Covaris E210 and purified them with AMPure beads, as instructed by the SureSelect XT protocol. I measured the samples on the Bioanalyzer 2100 immediately following clean-up and received good peaks at the right area. However, I re-measured the samples as a positive control and I see that even though the peaks are still present for my sample, they are much shorter and it appears I have lost some gDNA.

My first instinct is to consider this nuclease contamination, but if this were the case, wouldn't I not see any evidence of DNA on the Bioanalyzer? I'm also wondering if I lost DNA following the freeze-thaw cycle the samples underwent, for they were stored at -20 degrees Celsius. Does anyone else have any ideas about what could be causing this DNA loss, as well as any suggestions about how I should prevent it? Thanks!

ECO · 02-24-2012, 01:16 PM

What's the original sample source (organism and if applicable, tissue) and purification method? How much shorter is shorter? Did the whole peak move

Nuclease contamination is a possibility. Freeze-thaw problems are very unlikely in my experience, especially if you're observing a dramatic change in sample quality from a single cycle.

I always suspect the Bioanalyzer. =] Any quantitation method that relies on pipetting of 1ul is by definition a ballpark method (unless you run several dilutions/reps etc).

moedog45 · 02-25-2012, 06:59 AM

The original source was tissue, and the DNA was purified with Agencourt AMPure XP magnetic beads. The peak is shorter by about 15 fluorescence units, but the peak itself did not move.

I talked to tech support about this and they told me if there is only one visible peak and the peak size hasn't moved to a smaller size range, then I shouldn't worry about nuclease contamination. Tech support then validated your opinion, ECO, and said it was likely discrepancy between the two reads. I guess storing nucleic material in water for a long time rather than a buffer makes me a bit queasy.

Thanks for the feedback!

vtosha · 02-29-2012, 04:03 AM

Do you measure concentration of your samples with another technics? Bioanalyzer not reliable for measuring of concentration.

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02-24-2012, 12:47 PM	#1
moedog45 Junior Member Location: Washingto, D.C. Join Date: Feb 2012 Posts: 4	DNA loss on Bioanalyzer About two weeks ago I sheared some gDNA samples on the Covaris E210 and purified them with AMPure beads, as instructed by the SureSelect XT protocol. I measured the samples on the Bioanalyzer 2100 immediately following clean-up and received good peaks at the right area. However, I re-measured the samples as a positive control and I see that even though the peaks are still present for my sample, they are much shorter and it appears I have lost some gDNA. My first instinct is to consider this nuclease contamination, but if this were the case, wouldn't I not see any evidence of DNA on the Bioanalyzer? I'm also wondering if I lost DNA following the freeze-thaw cycle the samples underwent, for they were stored at -20 degrees Celsius. Does anyone else have any ideas about what could be causing this DNA loss, as well as any suggestions about how I should prevent it? Thanks!

02-24-2012, 01:16 PM	#2
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	What's the original sample source (organism and if applicable, tissue) and purification method? How much shorter is shorter? Did the whole peak move Nuclease contamination is a possibility. Freeze-thaw problems are very unlikely in my experience, especially if you're observing a dramatic change in sample quality from a single cycle. I always suspect the Bioanalyzer. =] Any quantitation method that relies on pipetting of 1ul is by definition a ballpark method (unless you run several dilutions/reps etc).

02-25-2012, 06:59 AM	#3
moedog45 Junior Member Location: Washingto, D.C. Join Date: Feb 2012 Posts: 4	The original source was tissue, and the DNA was purified with Agencourt AMPure XP magnetic beads. The peak is shorter by about 15 fluorescence units, but the peak itself did not move. I talked to tech support about this and they told me if there is only one visible peak and the peak size hasn't moved to a smaller size range, then I shouldn't worry about nuclease contamination. Tech support then validated your opinion, ECO, and said it was likely discrepancy between the two reads. I guess storing nucleic material in water for a long time rather than a buffer makes me a bit queasy. Thanks for the feedback!

02-29-2012, 04:03 AM	#4
vtosha Member Location: Moscow Join Date: May 2010 Posts: 36	Do you measure concentration of your samples with another technics? Bioanalyzer not reliable for measuring of concentration.