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Old 03-02-2012, 02:07 PM   #1
schizosaccharomyces
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Unhappy pico green discrepancies

Hello!

I have submitted genomic DNA for WGS to our core sequencing facility. The nanodrop reading says my DNA is ~50ng/microliter, but the pico green (which I am told is specific for dsDNA) is much lower. The core facility tech suggests that my DNA may be ssDNA, but I don't know why it would be ssDNA. I prepared the DNA using phenol chloroform extraction followed by EtOH precipitation. The DNA was resuspended in molecular grade water at 4 degrees for 24 hrs. The DNA looks good on a gel (it runs at >12kb fragments) and DNA prepared with the same protocol is cut with restriction enzymes (which cut only dsDNA).

I don't know what the problem with my DNA is and if I should tell the technician to proceed to library prep with my samples or not.

Please help me anonymous smart people!

Thanks
SaraH
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Old 03-02-2012, 03:03 PM   #2
ajthomas
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Quantification by UV spectroscopy is not very accurate. Fluorometry is much more accurate and that is why NGS protocols generally specify using it rather than UV spectroscopy. If I had to guess about your specific sample, my first thought would be that you may have a significant amount of RNA in your sample, thereby increasing the apparent concentration on the nanodrop, but not with Picogreen.
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Old 03-05-2012, 07:41 AM   #3
pmiguel
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Your DNA is not single-stranded, it most likely either:

(1) RNA. After RNAse treatment the RNA will fragment into very small oligos that you will not be able to visualize on your gel. But the source of absorbance of UV is the base, not the intact phosphoribose backbone.

Hence, degraded RNA absorbs UV just as strongly as non-degraded RNA!

Let's say that ethanol precipitation will remove about 90% of the RNA oligos. Since cells will have at least 100x more RNA than DNA, you will still have a 90% oligoribonucleotide solution as far as UV spec is concerned. (And, my experience is that is about what one sees with the average genomic "DNA" prep.

(2) Phenol absorbs strongly at 260nm! How strongly? Well a 0.1% solution of melted phenol in pure water fooled my nanodrop into thinking it was looking at a 500 ng/ul solution of DNA

Phenol's lambda max is around 270nm. So if you calculate 260/270 and that is lower than 1.2 or so, then you probably have a little bit of residual phenol that is throwing off your assay.

As ajthomas mentions above picogreen fluorometry is insensitive to either of these common contaminants of DNA preps.

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Phillip
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Old 10-17-2013, 06:37 AM   #4
Baoqing
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This response seems late, I had the exact same problem. I run a control of samples with RNA cleaned and uncleaned samples with picogreen. The untreated RNA samples reading was shooting to the roof. This is very different from what i had read on picogreen is not sensitive to the RNA presence. However, i might be wrong, because maybe there is a RNA concentration threshold to be sensitive to picogreen? it might happened to be that my untreated RNA sample, RNA concentration is too high?
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Old 10-17-2013, 08:34 AM   #5
pmiguel
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Quote:
Originally Posted by Baoqing View Post
This response seems late, I had the exact same problem. I run a control of samples with RNA cleaned and uncleaned samples with picogreen. The untreated RNA samples reading was shooting to the roof. This is very different from what i had read on picogreen is not sensitive to the RNA presence. However, i might be wrong, because maybe there is a RNA concentration threshold to be sensitive to picogreen? it might happened to be that my untreated RNA sample, RNA concentration is too high?
Here is the specificity picture they provide on their website:



That said, I would guess a typical cell as 1000x more RNA than DNA in it. So even at a low level of RNA sensitivity, a fluor might report substantially high before removing the RNA.

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Old 10-17-2013, 08:37 AM   #6
pmiguel
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Alternatively, maybe the double stranded stretches of the rRNA would fluoresce in the presence of picogreen.

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