[kerplunk412]

Mass of genome in one human cell is 6.6 pg, so in a DNA solution of 10 ng/ul we would have equivalent of DNA from 1515 cells which would be 45,450,000 fragments of 100kb. Most standard extraction methods will result in fragments less than 100kb. So, I do not see how one can justify that 45.5 million fragments in 1ul will aggregate in a solution to give 10x variation in consecutive reads. I think just a gentle flick would be enough to have a homogenous solution (if sample was frozen) and vortexing definitely would damage large DNA fragments.

Your logic makes sense to me, so maybe the difference in size before and after vortexing does not explain my observations. However, I tested this fairly rigorously and a few of my colleagues have tried this as well, so I can say with confidence that with the gDNA samples I was working with a gentle flick was not enough to get a consistent reading, vortexing was required. As far as damaging the DNA, I am pretty sure 10 seconds of vortexing will not cause enough DNA fragmentation to matter for most NGS applications. If it was that easy to fragment DNA into small pieces no one would need to buy a Covaris!

Edit: I should also mention that the variation seen before vortexing was at most about 2x. Variation after vortexing was ~1%.

[Terminator]

Linear detection range of PicoGreen is four orders of magnitude in 1 ng/ml to 1000ng/ml DNA concentration. As far as one calibrates fluorometer at 0 and 1000 range there is no need to any other concentration in between or standard curve. It seems to be waste of money and time. With correct calibration one needs only to multiply the fluorescence value in dilution factor to calculate original concentration of DNA sample.

The 500 reaction kit is cheap (60 cents/sample) and it takes a matter of minutes to add a few extra standards. You are correct regarding the linear detection range; however, I think you should review linear regression.

[nucacidhunter]

I have noticed something similar when quantifying gDNA by Nanodrop. What I saw was values that would vary quite a bit when reading different "drops" from the same tube of gDNA. This was solved by vortexing the DNA for 10 seconds. The idea is that the genomic DNA molecules are so large that one microliter might have varying amount of these large DNA molecules. Following vortexing the gDNA is in much smaller fragments, which allows it to exist more evenly in solution such that every microliter will have a much more similar amount of DNA. Think of it of grabbing handfuls of sand versus handfuls of medium sized rocks and weighing them. The handful of sand will be very close to the same weight each time, but the rocks will vary much more. I haven't tested the theory about large DNA vs sheared DNA, but we have tested vortexing DNA for 10 seconds prior to reading on the Nanodrop and it definitely results in much more consistent readings.

Mass of genome in one human cell is 6.6 pg, so in a DNA solution of 10 ng/ul we would have equivalent of DNA from 1515 cells which would be 45,450,000 fragments of 100kb. Most standard extraction methods will result in fragments less than 100kb. So, I do not see how one can justify that 45.5 million fragments in 1ul will aggregate in a solution to give 10x variation in consecutive reads. I think just a gentle flick would be enough to have a homogenous solution (if sample was frozen) and vortexing definitely would damage large DNA fragments.

[nucacidhunter]

I'm not sure why the Qubit only requires two standards (seems crazy).

Linear detection range of PicoGreen is four orders of magnitude in 1 ng/ml to 1000ng/ml DNA concentration. As far as one calibrates fluorometer at 0 and 1000 range there is no need to any other concentration in between or standard curve. It seems to be waste of money and time. With correct calibration one needs only to multiply the fluorescence value in dilution factor to calculate original concentration of DNA sample.

[kerplunk412]

I have noticed something similar when quantifying gDNA by Nanodrop. What I saw was values that would vary quite a bit when reading different "drops" from the same tube of gDNA. This was solved by vortexing the DNA for 10 seconds. The idea is that the genomic DNA molecules are so large that one microliter might have varying amount of these large DNA molecules. Following vortexing the gDNA is in much smaller fragments, which allows it to exist more evenly in solution such that every microliter will have a much more similar amount of DNA. Think of it of grabbing handfuls of sand versus handfuls of medium sized rocks and weighing them. The handful of sand will be very close to the same weight each time, but the rocks will vary much more. I haven't tested the theory about large DNA vs sheared DNA, but we have tested vortexing DNA for 10 seconds prior to reading on the Nanodrop and it definitely results in much more consistent readings.

[Terminator]

I did run undiluted controls a couple of times but should start doing it every time. If I run undiluted controls over and over (without changing anything) there variation of maybe around 5–20% or so (which I have no problem with, at least it's qualitatively similar). The larger error comes in when we change the dilution and especially when we concentrate the DNA to a lower volume but then it is more viscous—so I imagine as suggested above that this is a serious source of error.

That is a great idea to do the extra standards across the range and then plot- thanks!

I'm not sure why the Qubit only requires two standards (seems crazy).

I found a post (I don't recall the specific thread) where a user recommended running larger volumes for dilute DNA samples. This may also be worth a shot for reducing variability.

Best of luck!

[Myrmex]

Hello,

Did you re-run the undiluted DNA side by side with the diluted DNA as a control?

What do the raw fluorescence readings for controls look like between the two runs?

Assuming you have a homogeneous mixture of DNA with no clumping, try adding additional Qubit standards. I have seen a fair deal of variation in the past so now I run 10 controls (2 x each of 0, 25, 50, 75, and 100 ng) and plot it myself to calculate my DNA concentrations.

I did run undiluted controls a couple of times but should start doing it every time. If I run undiluted controls over and over (without changing anything) there variation of maybe around 5–20% or so (which I have no problem with, at least it's qualitatively similar). The larger error comes in when we change the dilution and especially when we concentrate the DNA to a lower volume but then it is more viscous—so I imagine as suggested above that this is a serious source of error.

That is a great idea to do the extra standards across the range and then plot- thanks!

[Terminator]

Hello,

Did you re-run the undiluted DNA side by side with the diluted DNA as a control?

What do the raw fluorescence readings for controls look like between the two runs?

Assuming you have a homogeneous mixture of DNA with no clumping, try adding additional Qubit standards. I have seen a fair deal of variation in the past so now I run 10 controls (2 x each of 0, 25, 50, 75, and 100 ng) and plot it myself to calculate my DNA concentrations.

[Myrmex]

Okay, that makes a lot of sense. Thanks so much for your help everyone, we've been struggling with this for a while. It's nice to have something else to go off of.

[nucacidhunter]

In this case you need to do clean up only as DNA have already been extracted. Sample loss depends on column specifications but one can maximise recovery by eluting with hot buffer and double elution. In my experience 10-15% loss is normal. In your case it will depend on how strongly DNA is bound to viscous material as well which may go through column. Best approach is to trial with less precious sample by quantifying with dsDNA specific reagents before and after cleans up because dsDNA will contribute to final library.

[Myrmex]

okay thank you very much—I will go from there... Do you have any feeling for whether or not I should be concerned about DNA loss with filter extraction, or filter cleanups? I know that is a common fear with filters but don't know how founded it is. We have been told by the manufacturer that there should be 95% recovery unless pieces are over 50kb. I have no idea if we have pieces of DNA that big or not. I assume that I am probably overly-fearful since I imagine that others have done GBS/RADseq protocols with filter extractions of DNA...

[nucacidhunter]

One way to get rid of viscous material is column clean-up. In this case sample need to be diluted before addition of binding buffer (as much as kit binding buffer volumes allows) to prevent clogging columns. In addition DNA concentration can be adjusted by elution volume.

[Myrmex]

I can think of three reasons:

1- Pipetting inaccuracy caused by calibration/maintenance issues or operator
2- Presence of viscous material in DNA preps (mostly in non-column based extractions). This would show as streaks in wells of gel. This can be avoided by spinning DNA tube or plate at high speed for 5 min to precipitate non-soluble materials and transferring from top of wells.
3- Proper mixing of DNA with reagents or after dilution

Okay, thank you, this makes a lot of sense. We do have viscous material in our CTAB extractions which is one of the ideas we came up with for why this is happening. When I decrease the volume of the sample in a SpeedVac from 100µl to 50µl the material gets a lot more viscous. I have been trying to mix things very well before measuring on the Qbit but now I realize that maybe I should be spinning them down first and taking only the water liquid on the top.

The other option I guess is to re-extract with a column extraction- I was just worried about getting enough DNA, as we are already riding a thin line far as that is concerned...

[nucacidhunter]

I can think of three reasons:

1- Pipetting inaccuracy caused by calibration/maintenance issues or operator
2- Presence of viscous material in DNA preps (mostly in non-column based extractions). This would show as streaks in wells of gel. This can be avoided by spinning DNA tube or plate at high speed for 5 min to precipitate non-soluble materials and transferring from top of wells.
3- Proper mixing of DNA with reagents or after dilution

[SNPsaurus]

There are very careful ways to do it, and there are ladders to help estimate the amounts, but you can just run out an agarose gel and see what you see, and in this case that would help.