DNA quantification instruments

[jarlemag]

Hello. As mentioned in a few previous posts I'm shopping for lab equipment, and one of the pieces I'm missing is some kind of equipment for quantifying DNA. I have considered several alternatives, and would like to ask about your experiences. To specify my needs a bit more, I am primarily interested in doing DNA concentration measurements of samples (PCR products) to be sent for sequencing. So preferably, the method should not require a too large sample volume.

Previously, I have used solely the NanoDrop (ND-1000) for measuring DNA concentrations. It's a nifty instrument and I'd love to have one but the prices I have seen have been in the $10 000 range, which is far above my current budget. Because it is a relatively new product, there are also few used units being sold. The main attraction of the NanoDrop is the microliter sample volume and ease of use. The main criticism I have seen of the ND, and which I agree with, is poor accuracy at low DNA concentrations with too high DNA concentrations being reported. However, the absorbtion curve being presented visually for each measurement at least makes it possible to evaluate how much trust to put in each measurement. There are several NanoDrop "clones" available too, such as Quawell (http://www.quawell.com/), but these are still priced at $5000 or so, and I'm unsure of the quality.

Although I'm unlikely to purchase one in the near future, I'd still like to hear your opinions on NanoDrop and other microliter-spectrometers. Are they worth the expense?

An instrument which I have seen touted as a cheaper and more accurate alternative to the NanoDrop is the Qubit Fluorometer.(http://www.lifetechnologies.com/no/en/home/brands/product-brand/qubit.html?cid=fl-qubit). Especially at low DNA concentrations, the Qubit seems to be more useful. The downsides are larger sample volume (up to about 20 uL needed), some sample preparation needed, and the need to buy instrument-specific consumables. Still, this instrument can be had for about 2-3000 $USD, which is affordable and would seem to give good value if the Qubit is as accurate as the advertisments suggests.

Have any of you used the Qubit or similar fluorescent-based DNA quantification instruments? What has your experience been?

The third alternative I am aware of is a plain old spectrometer. I also want something for measuring optical density of bacterial cultures, so a spectrometer is something I'd like or need for that purpose anyway. My main concern with using one for DNA concentration measurements is the need for (much) larger sample volumes than with NanoDrop or Qubit. Rather than buying a large, heavy and expensive all-round spectrometer, I am considering buying an Eppendorf Biophotometer (http://www.eppendorf.com/int/index.php?pb=63cfedee26795e18&action=products&contentid=1&catalognode=53772) (That's the newest model, but there are used older models for sale much cheaper). This seems to give a good all-round package for OD600, DNA/RNA and protein concentrations at smaller size and lower price. There are also small, cheap generalized spectrometers of Chinese make available, but I am very unsure of the quality of these...

Do you any of you have experience with the Eppendorf Biophotometer/Biospectrometer series or other spectrometers for measuring DNA concentrations? How much work goes into making standard curves and calibration? What is the minimum practical sample amount?

And finally, for those who have used several of the methods above: What do you recommend as the "go to" method for measuring DNA concentrations for a new DIYBio laboratory?

Looking forward to hear your opinions!

Best regards,

Jarle Pahr

[Nathan McCorkle]

The nanodrop /is/ just a plain old spectrometer, with two fiber optic cables leading to and from the microdrop stage. Search Google for TakeItApart nanodrop to see pics of the inside.

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[Sebastian Cocioba]

I own a qubit and its indispensible. The strand specific DNA stain coupled with the tiny footprint makes it a fantastic deal. Only catch is that it won't tell you things like salt conc. so you can't tell how pure your sample is.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

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From: Nathan McCorkle
Sent: ‎7/‎31/‎2014 11:30 AM
To: diybio
Subject: Re: [DIYbio] DNA quantification instruments

To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/CA%2B82U9LQ6dW%3DoPJpHeNaqt75VJ4ZHOWWkhEVOLqZN--5uDMsmA%40mail.gmail.com.

[Cory Tobin]

I recently bought an old Eppendorf Biophotometer. I have access to a
Nanodrop at my university's campus and the Biophotometer reports DNA
concentrations within one 1 or 2 ng/uL of what the Nanodrop reports.
There are a few downsides to the Biophotometer, though.

1) It only reads at fixed wavelengths (230,260,280 and a few others)
so you can't get a full spectrum measurement like on the Nanodrop.

2) By itself it does not handle microliter volumes. It takes a
cuvette like usual. But it can also handle the Hellma TrayCell or the
Eppendorf uCuvette G1.0. Those are sold separately but allow for
using 1-2uL samples. These devices fit into the cuvette receptacle
and focus the light through a small point where the sample sits. I
have a Hellma TrayCell and it works just fine. It's a little more of
a pain to clean out the old sample compared to the Nanodrop but I
don't mind.

3) It doesn't report data to a computer so you have to resort to
recording your data into your notebook with a pen. I don't mind but I
know this would be a deal breaker for some of my colleagues.

4) The TrayCells are hard to find (occasionally one will show up on
ebay) and the uCuvette G1.0 costs around ~2,000 USD.

The positives are

1) Way cheaper than a Nanodrop.

2) Works just as well as a Nanodrop.

3) Can also do OD measurements so you don't need a separate device.

-cory

[John Griessen]

On 07/31/2014 05:52 PM, Cory Tobin wrote:
> It doesn't report data to a computer so you have to resort to
> recording your data into your notebook with a pen. I don't mind but I
> know this would be a deal breaker for some of my colleagues.

What format of data do your colleagues want? Or, (even worse), is there a specific
vendor and app they use?

[Cory Tobin]

On Thu, Jul 31, 2014 at 5:10 PM, John Griessen <jo...@industromatic.com> wrote:
> What format of data do your colleagues want? Or, (even worse), is there a
> specific
> vendor and app they use?

The Nanodrop software can export your data as an Excel spreadsheet. I
know plenty of people who would whine for days if this was no longer
an option. Not because Excel is particularly useful but because
that's the way they've done it for years. Some biologists are
obstinate.

[Nathan McCorkle]

Excel read/write in python is easy.

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[Nathan McCorkle]

GUIs in python are pretty easy too. Here's some old code I wrote to plot spectrometer data in python:
https://github.com/nmz787/open-spectrometer/blob/master/desktop-software/openSpectrometer.py

I've been working with wxpython lately for GUIs, and would have to look at the pygtk forums and bug tracker to see if it would be worth switching to wxpython or not in any future updates/forks of this. I kind of forgot about it when I was looking at GUI toolkits a few months ago.

[Cathal Garvey]

You mean via CSV or is there a direct-to-excel module out there? I ask
because, though I loathe all things WinMac/Silo'd/Hopelessly-broken, a
colleague has asked me to provide him an Excel compatible interface to
HMDB and I've found the CSV standard library module to be insufficient
for the task; so, I'm writing a strictly typed schema-based CSV writer.

If there's a direct-to-excel method out there that's better than
Python's standard csv module, it might save me a lot of time..

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[W. Estell]

You should be able to get by just fine using your ladder for quantification.

[Nathan McCorkle]

There are a few direct to excel libraries. I haven't used them lately,
and can't remember which exactly I've used... you'll have to spend a
few minutes looking to see which have the most bug reports open, or
how active the user message boards are... or just try a few and see
which one works best for you.

--
-Nathan

[Josiah Zayner]

With different cuvettes a spectrophotometer can do very small sample volumes. Most specs use small beams so you can put the cuvettes on a platform to decrease the amount of sample needed. Also, you can always just dilute your sample to increase the volume and unless it is a really low concentration you shouldn't have many problems. I guess it all comes down to how much money you want.have to spend.

[SC]

Hi Jarlemag,

 

You don't need a piece of equipment like that for setting up sequencing samples.  You can run a gel with a 1 kb ladder marker, and eyeball the comparison of band brightness.  That's good enough, and you can save your money for other things.

 

Good luck!

Stacy