TITLE: [DIY Microliter Fluorometer]
[As any budding biohacker eventually figures out, processes such as PCR and DNA sequencing are subject to the GIGO principle: Garbage-In-Garbage-Out. For example, if the starting DNA concentration in your sample is too low, the results of a PCR or sequencing run will be crap, or not very informative. That’s why it’s useful to measure the DNA concentration in your sample before anything else. One way to do this is to measure the level of fluorescence when you mix your sample with a DNA dye. The device to do this measurement is called a fluorometer. Such devices are relatively simple, but can be expensive when bought commercially. That means: DIY time! The idea is to use LEDs (to excite the dye) and a sensitive light sensor (to measure the fluorescence). The sample volume needed should be low (microliters).]
- [Light sensor - check, I’ll bring two TSL235R and two TSL2591 sensors
- LEDs - check, I’ll bring a few blue LEDs with known suitable emission spectra
- optical filter - somewhat check, I’ll bring a small square of a Knight Optical filter and a sample set of photography filters
- possibly other optical elements like lenses?
- DNA dye - check, I’ll bring some GelGreen
- DNA samples - check, I’ll bring some DNA ladders w/o loading dye that we can use to make dilution series with known DNA concentrations
- sensor/sample holder contraption thingy
- Microliter pipettes + tips - check]
LINKS TO MORE INFORMATION
Please add constructive feedback, suggestions, offers of equipment or skills, and gestures of wild enthusiasm as replies below. Thanks!
Very nice idea - I like it a lot!
I know that somewhere out there in the web, there is a collection of building instructions for various wetlab equipment, but I can’t find it any more … (sadly, I never had too much contact with that before).
It seems like you already have all molecular biology equipment and samples, so I don’t have to try “stealing” anything from my workplace ^^
I have one question though: is it technically to challenging to aim for UV absorption spectra of DNA (at 280 / 260 nm), or is there another reason you are aiming for dye dependent fluorescence?
Since I’m not sure if protective gloves (Latex or similar material) will be available in general, I’d like to mention that in regard to the required equipment.
Well, if you can bring small quantities of other fluorescent dyes in the same spectrum (excitation around ~490nm, fluorescence around ~520, like SybrGreen or PicoGreen or MidoriGreen), that could be interesting, to compare different dyes, see whether there are any that work better than others for this purpose…
And good point on the gloves! I’ll bring some nitrile ones. I’m personally not too worried about the modern DNA dyes, but better safe than sorry.
Concerning the deep UV thing: I’d say it’s not really technically more challenging, but logistically. That means: I have a bunch of cyan LEDs and visible-spectrum light sensors, but I don’t know how the light sensors I have would perform for deep UV, and I don’t have any deep UV LEDs lying around. They’re also quite a bit more expensive, though affordable at this point. (Full-spectrum light sources are too bulky for my taste.) I thought about also ordering some deep UV LEDs before the SHD, but they would ship in from China and there’s no way they’re gonna get here in time.
Eventually, I’d also like to build a microliter volume UV spectrophotometer, though Basically, I’d like to have both, since they have slightly different use cases and limitations. Quote:
“Spectrophotmetric methods do not measure nucleic acids directly, but the absorbance of whatever is in your tube. We use the qubit for measuring dsDNA, which it does by measuring the fluorescence of a double stranded DNA specific dye. Therefore, if there is protein, RNA or nucleotides in the sample, they are not going to significantly contribute to the measurement as they would with the nano drop.
We have found up to a 10-fold overestimation using the NanoDrop. That is not really a problem for cloning for example, but for NGS, it can be a critical and very expensive difference. Moreover, I wouldn’t trust the Nanodrop at all under 10 ng/ul, where the Qubit handles sub-ng/ul fine.
Both machines have their place in a lab, but for DNA concentration, i’d go for the Qubit.”
I’d still be careful about anything that sticks to DNA. Ethidium bromide’s toxicity is debated, but for the alternatives often the only source is the manufacturer’s data sheet, but that’s only a word of caution. I’m sure you know how to handle the stuff.
The biggest challenge I see with fluorescence devices is the filtering of excitation and emission wavelengths. A cool thing I have seen somewhere (great sources, I know) is using the grid of a CD as a refraction grid to make a light spectrum. By turning the grid you can scan the spectrum over a photosensor and get a full spectrum of wavelengths and pick out the relevant ones (depending on calibration).
I love the whole project idea, I think taking simple optoelectronic lab equipment into the DIY field is great and super useful.
Ok, I hear ya. So, in the interest of safety first: I’ll bring nitrile gloves, make sure that anyone who wants to handle the dyes knows the risks and encourage them to wear gloves as well
Using CDs as a refraction grid sounds like Hackteria… Was this the one you were thinking of? https://hackteria.org/wiki/index.php/DIY_NanoDrop
I was hoping that for the fluorescence approach, we could get away with using suitable LEDs and a long-pass filter. This is the one I’ll be bringing: https://www.knightoptical.com/stock/optical-components/uvvisnir-optics/filters/long-pass-filters/acrylic-longpass-filters/colour-filter-acrylic-type-50x50mm-510nm-long-pass/
My own lab actually uses EtBr instead of dyes, but I can still try to ask around if anyone else has something they could donate.
You’re of course right, that the methods based on fluorescent dyes (like qubit) are more sensitive and less prone to be affected by contaminations, but they also require the continuous use of the dyes, which makes them more expensive in the long run. That’s why I’m mainly using UV based measurements, except for very ‘important’ samples.
I don’t have enough technical expertise to say anything about filters vs refraction grids, but it looks like it should be possible to try both?
+1 for gloves and safety
Here’s an interesting link: http://blogs.sciencemag.org/pipeline/archives/2016/04/18/the-myth-of-ethidium-bromide
Science is really governed just as much by emotion, superstition, and cultural expectations as any other human endeavour.
Lab Safety is my homeboy!