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UV Transilluminator

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UV-transilluminators are used in molecular biology labs to view DNA (or RNA) that has been separated by electrophoresis through an agarose gel. During or immediately after electrophoresis, the agarose gel is stained with a fluorescent dye which binds to nucleic acid. Exposing the stained gel to a UVB light source causes the DNA/dye to fluoresce and become visible. This technique is used wherever the researcher needs to be able to view their sample, for example sizing a PCR product, purifying DNA segment after a restriction enzyme digest, quantifying DNA or verifying RNA integrity after extraction.

This Instructable tutorial describes how to make a UVB (310nm) transilluminator with a 7 x 7 cm window for viewing ethidium bromide (or SYBR-Safe) stained DNA mini-gels. Once all of the materials are collected, the actual assembly time is approx. 1-2 hours. Some soldering is required.

Safety Notes:
  1. Because ethidium bromide is a toxic chemical with strict safety protocols, it is only recommended that you use this dye in a lab with established handling, storage and waste disposal procedures in place. Other users are strongly recommended to use SYBR-Safe instead, which can be handled and disposed of more safely.
  2. The transilluminator does come with a safety lid for viewing the gel. However, when the lid is not in place, safety glasses must be worn when operating the UVB bulb.
  3. If you prefer to avoid UVB altogether, we can recommend the blue light LED transilluminators such as the one described in this instructable instead.
 
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nthamaragsa25 days ago

Hi, Jorodeo
Where I buy U325C glass ?, Because of in Thailand not have it.

macowell2 years ago
Terrific instructable! This looks like it works. The sub-$100 transilluminator is a milestone. Great job in particular explaining how to use the professional-grade 312nm UVB bulb and ballast (only $28 for the bulb! awesome! Cat# 11-2122 at http://www.fotodyne.com/content/trans_access).

I have experimented with making my own transilluminator for GelGreen and SybrSafe stains, similar to the Pearl Biotech transilluminator instructable you linked to. I have found that 2424 Blue Acrylic works as prefilter and 2422 Orange Acrylic works as a postfilter for those stains. I got sample sheets from ridout plastics.  Attached is a photo of a gelgreen-stained dye sandwiched between these two acrylics illuminated underneath via a blue LED array.

Any interest in working on a design for open-source reusable gel cassettes for making precast gels?  It would be possible to create these cassettes directly out of the filtering acrylics.
2011-09-21_GelGreen-RidoutPlastics-2424_2422.jpeg

is this acrylic sheet are completely dull or light transmitting?

jorodeo (author)  macowell2 years ago
Thanks for your comments. I actually had a lot of fun building it once I was able to source all the parts. I've used commercial UV transilluminators for years and they are so expensive ! Your version with leds and blue/orange acrylic filters looks like it works well too. I have toyed with the idea of having a second set of parts which would convert the UV transilluminator into a blue/orange version. In theory I guess you could swap the UV bulb for the white light version ($15 from fotodyne) and instead of the transilluminator glass use the blue acrylic, and then make the lid from orange acrylic ? I should try that. Thanks for sharing your acrylic sources and sample gel image.

I would be interested in the gel cassettes. Have you get some ideas for this project already ?
Please excuse my naivete but you've chosen the filter because it transmits wavelengths in the absorption range of the ethidium bromide (around 300nm) and also transmits in the emission range of the ethidium bromide (around 700nm) as well, correct?

In the picture above, were the bands visible to the naked eye or did you have to use a UV camera to get them to show up?

Also, does this transilluminator fit under your (mini) gel electrophoresis system so that you can see the bands while the electrophoresis is running?

Sorry for the deluge of questions, thanks again for the great instructables!
jorodeo (author)  dorkusmonkey2 years ago
Hi dorkusmonkey,
The U325C transilluminator filter glass from HOYA optics is pretty essential for viewing the DNA bands, at least with this UVB lamp setup. It is a UV transmitting, visible light absorbing filter. So the only light coming through the filter to the gel sitting above it is the UV light. This filter does not interfere with the ethidium bromide/DNA fluorescing bands, so we don't have to worry about that. You can indeed see the bands with the naked eye - but with the UV protective goggles or safety lid in place of course !
The transilluminator doesn't fit under the gel electrophoresis tank. I did plan to design it that way initially, but it just didn't work out. Maybe in the future ......
I have attached a pdf document from HOYA for the U325C filter for your interest.
Thanks !
I should have also mentioned, I found an excitation/emision spectra for ethidium bromide/DNA here:

http://www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/Product-Spectra.1305dna.html
ktkeith2 years ago
What is the purpose of the transilluminator glass? Is it polarized? Since you are laying the gel directly on the solacryl sheet, why do you need the transilluminator glass and the two lower cover layers to hold it in place?

Note also: the supply link you give for the transilluminator glass - hoyafilters.com - redirects to micro-tools.com, which does not carry Hoya Filters products or, apparently, have anything similar to this. Again, is it necessary?
jorodeo (author)  ktkeith2 years ago
Hi, thanks for your feedback ! I should have probably done a better job at explaining the purpose of some of these parts :)

The transilluminator glass is a very important part ! I found that out the hard way. Without the filter glass, it is very difficult (impossible for me anyway) to see the DNA bands through background light.

The solacryl cover protects the transilluminator glass from wear and tear, especially if you plan to cut out your DNA bands.

My apologies for the link - I have fixed it now. Should have been hoyafilter.com not hoyafilters.com.

jorodeo (author)  jorodeo2 years ago
As pointed out by dorkusmonkey below, I should have directed you to the hoya optics website: http://www.hoyaoptics.com/color_filter/index.htm.
Checking out hoyafilter.com I didn't find anything that matched the "U325C" product id. Calling around, they pointed me to hoyaoptics.com and I found a web page with what I believe is what you're using:

http://www.hoyaoptics.com/color_filter/uv_transmitting.htm

With the some specs on the tramsmittance etc. here:

http://www.hoyaoptics.com/pdf/U325C.pdf

Could speak a little bit on how you ordered the filter: i.e. if they'll sell to individuals in quantity 1 or whether you had to acquire it by some other means? How much was the filter?

Your transilluminator is beautiful by the way!
jorodeo (author)  dorkusmonkey2 years ago
Hi, yes that is the one, Hoya Optics ! I will fix the link above. Sorry you had to dig around to find the information. I basically used the contact form on their website to request a quote for the U325C glass. The glass is cut to your custom size, at least it was for me anyway, and cost $40 per 70x70mm part. However, they had a minimum order of at least 3 parts, so really it was $120 for the 3 pieces. Let me know if you have any other questions about this .... I'd be happy to help !
Exocetid2 years ago
OK, when I first saw this (by title and final pic only) I thought, "What's this for?" and then seeing the description realized what it is. My son does a lot of Western Blots in his cancer research work, so I imagine he has access to a research grade one in the lab--I'll have to ask him, but I digress.

My idea is that this would make a great display case/terrarium for a scorpion or other UV-luminescent creature!
UVB kills stuff. A regular "blacklight" bulb that you can find in party stores would be better for a scorpion.

Western blots are usually treated with an antibody labelled with an enzyme and read by one of two ways 1) add luminol and the enzyme makes it glow, you take a picture with a sensitive camera or you put in on a film OR 2) add a chemical that the enzyme makes change colour, then you can take a regular photo. Usually people won't use fluorescent antibodies, as far as I know.
UVB starts at about 290 nm. You may want to review Kloock's short communication in the Journal of Arachnology (37:368–370) Reducing scorpion fluorescence via prolonged exposure to ultraviolet light. He exposed them to 290–390 nm for close to a month.

I have no idea what gets used in a Western Blot, but my son uses them extensively and has also done electrophoresis using fluorescence techniques--I'm just say'in.
My mistake, sorry. I didn't read the details properly and got mixed up and was thinking about sterilising UV (UVC).

It's still kind of small for a scorpion to live in though, unless you're keeping a very small species as a pet ;)
I think you are right, too small and not nice for a pet.

I wasn't paying much attention to scale when I thought of a scorpion display case. Of course, in retrospect someone could build this as a scorpion terrarium, just make the box on top larger. Put some fluorescent rocks in, etc. Would be impressive. One could also have the UV lamp on a timer given that they are pricy, or a three way switch with off, UV and visible lamps.
Thetis2 years ago
Gosh, this takes me back to youthful lab days. We did a lot of Southern Blotting but had to don a protective mask and use a UV lamp in a darkroom to look at our gels and extractions.

How I miss the smell of Mercaptoethanol in the mornings.. : P