Frequently Asked Questions
Taken verbatim (with only minor editing for brevity) from various conversations and correspondence. Keep them coming!

FAQ - Dark Reader Components

Do we have to use the amber glasses for eye protection?
What is the actual size of the blue light area of the DR46 vs the outside dimensions of the entire box?
How resistant is the blue screen to scratching?
Which has higher intensity, the DR46 or DR88?
How long do the LEDs last?

FAQ - Imaging
I would like to know which digital cameras work with the Dark Reader.
I need to know if the DR88 transilluminator will work with our 'XYZ' imager.
We have a camera for EtBr gels using a UV transilluminator. Do I need a new filter?
Our photographs are coming out fuzzy.
I would like to buy a Dark Reader camera filter for my 'XYZ' camera. Which filter fits?

FAQ - DNA & Protein Stains
How sensitive is the Dark Reader Transilluminator for DNA detection?
What is the cost of the new DNA dyes?
My gel is very thin & fragile. Can I view it on a glass plate on the Dark Reader ?
Why are my DNA lanes smeared?.
Will the Dark Reader work with EtBr or GelRed?

FAQ - More
I would like to know if the fluorophor XYZ is compatible with the Dark Reader.
Does the DR work with GFP?
Can the Dark Reader Spot Lamp be used to observe EYFP in plants?
Can the DR be used for oligonucleotide visualization in PAGE gel on fluorescent TLC plate?
How well does the Dark Reader perform with 96-well plates?
Why should I worry about UV Exposure? I use a UV box inside a hood with a CCD camera.

FAQ - Dark Reader Components

Do we have to use the amber glasses for eye protection?
The glasses are not for eye protection. (Indeed, do not use them with a UV unit because they do NOT provide protection against UV !)
The light from the Dark Reader is all visible blue. Of course, it is fairly bright so you should not look directly at it for too long (without the glasses or amber screen in place) - just like any other visible light source - but there is essentially zero UV in it. The function of the glasses is optical - they prevent the blue excitation light from swamping the much fainter fluorescent light from the sample. They have exactly the same optical properties as the amber screen and DR camera filters. The glasses are especially useful for when you are cutting bands out of a gel. They save you trying to work around the amber screen.

What is the actual size of the blue light area of the DR46 vs the outside dimensions of the entire box?
The viewing surface is 15 x 19 cm. The outer dimensions are only somewhat larger at 18.5 x 22.5 cm. Check out our web page www.clarechemical.com/transilluminator.htm for more physical dimensions on all our units.

How resistant is the blue screen to scratching?
The blue screen is made of plastic and will scratch more easily than a UV screen. However, this is not an important factor in performance of the Dark Reader - we have had a Dark Reader in use for several years in a cloning lab and it has been used (and abused) on a daily basis. Though the blue surface is somewhat scratched when viewed under normal lab lighting, in operation all but the most serious scratches disappear. To protect the blue screen while cutting out DNA bands an ordinary piece of glass can be placed under the gel without any loss in sensitivity. In addition, remember that we can replace a blue screen for about $175-250 whereas a new UV screen will cost hundreds of dollars more.

Also note that the blue screen is much more impact resistant than a typical UV screen: drop a camera on a Dark Reader and the blue screen will remain intact. (No guarantees on the camera.)

Which has higher intensity, the DR46 or DR88?
The DR46 and DR88 have almost identical light intensities. Besides, a more intense excitation is not necessarily going to help you detect smaller amounts of fluorescence. The detection level will be limited by the difference between the fluorescence signal from the sample and the gel background and that ratio will be the same whatever the excitation intensity.

How long do the LEDs last?
The LEDs used in the DR-46 and DR88 transilluminators are rated for 50,000 hours (over 17 years of 8 hours-a-day operation!) and the DR-195 lamps for 6,000 hours.


FAQ - Imaging
Here's a good 'rule of thumb': if you can see your sample fluorescence by eye, but not in your photograph, the problem is within the imaging setup!

I would like to know which digital cameras work with the Dark Reader.
Just about any digital camera can be used - I have heard of students using their cell phones to photograph gels. In general though, a digital camera with manual control of exposure time, focus and f-stop is prefered. Many researchers like the Canon G-series cameras. The current model is the G12.

I would like to know if the DR88 transilluminator will work with our 'XYZ' imager.
I am not familiar with the 'XYZ' system but the answer is: Yes, in theory.
In practise there are a couple of issues:
- the physical size of the cabinet - is it big enough for the DR88.
- the electrical power supply into the cabinet. The XYZ may use a different style plug so you may need an adapter. We have several styles available.

We have a camera for EtBr gels using a UV transilluminator. Do I need a new filter?
You should always use a Dark Reader filter - either the amber screen or a separate DR camera filter. This filter is optimized to work with the Dark Reader. An amber screen is included in the basic transilluminator package.

Our photographs are coming out fuzzy.
There are 2 possiblities:
- You may be getting condensation forming on the underside of the amber screen. This will happen if the gel is warm. The condensation is difficult to see directly but it does result in a fuzzy photo and a loss of sensitivity. The remedy is to use a separate DR camera filter or to simply put a piece of plastic wrap over the gel.
- If you are using the Polaroid camera attached to a hood on top of the amber screen, then increase the f-stop to 5.6. This will increase the depth of field sufficiently to bring the gel back into focus. Increase the exposure time to offset the smaller aperture.

I would like to buy a Dark Reader camera filter for my XYZ digital camera. Which filter fits?
This question should be addressed to whoever supplied the camera. The specifications for lots of cameras can be found on www.dpreview.com

Do you recommed a filter on the camera or to use directly the amber screen?
The optical properties of the amber screen and the DR camera filters are identical, but having a separate filter is usually going to be a more flexible approach.



FAQ - DNA & Protein Stains
How sensitive is the Dark Reader Transilluminator for DNA detection?
With SYBR Green, SYBR Gold and GelStar the detection limit, by eye, is 50 -100 pg of dsDNA. For ethidium bromide (EtBr) it is about 10 ng.

With a CCD camera system it is possible to detect 10 - 20 pg of SYBR-Green/Gold or GelStar-stained dsDNA.

The newest SYBR dye - SYBR Safe - is much less sensitive than Green or Gold. It is possible to detect about 1 ng by eye and 500 pg using a CCD camera.

What is the cost of the new DNA dyes?
The purchase price of the new DNA stains is significantly higher than that of EtBr. However, it is important to remember that, for example, SYBR Green is 5 -10 times more sensitive than EtBr and less mutagenic. Many people will consider these advantages sufficient to outweigh the additional cost.

Because the new generation of DNA stains are generally 5 - 10 times more sensitive than EtBr, it is possible to load correspondingly less DN Asize standards and PCR reactions. This can result in significant savings. For example, a typical mini-gel with 2 lanes of DNA standards costs a total of about $1.86 if stained with EtBr and $1.43 if stained with SYBR Gold. If the samples loaded onto the gel are PCR reaction products, the use of SYBR Gold can result in savings of over $10 compared with EtBr.

My gel is very thin & fragile. Can I view it on a glass plate on the Dark Reader?
Yes you can. No problem. Because the Dark Reader light is all visible blue, it passes easily through glass and plastic and there is virtually no loss of sensitivity. The Dark Reader also works very well for observing fluorophors in plastic tubes, Petri dishes, 96-well plates, etc.

Whay are my DNA lanes smeared?
Too much DNA! The GelStar + Dark Reader combo is at least 10 times as sensitive as the UV + EtBr method. So, if you are loading your ‘usual amount’ of DNA, you are going to be seeing all sorts of schmutz! Reduce the DNA loaded on the gel by a factor of 5 - 10.

Will the Dark Reader work with EtBr or GelRed?
Both EtBr and GelRed are UV stains and are always going to look better on a UV box. It is possible to see DNA on a Dark Reader, but it is never going to look very pretty. To maximize viewing with EtBr it is important to:
- view in a completely dark room and give your eyes a few moments to adjust.
- use a lower amount of EtBr (0.2 - 0.5 ug / mL)
- load plenty of DNA (20+ ng / band)


FAQ - More
I would like to know if the fluorophor 'XYZ' is compatible with the Dark Reader.
The ex/em maxima of XYZ are around 534/570. The excitation maximum would, at first glance, seem to be a bit on the long side. However, from looking at the spectrum there is a fair amount of excitation below 500 nm and this is what the Dark Reader would ‘go after’. Tetramethylrhodamine (546/576) is the dye with the closest spectral characteristics we have experience with, and this works surprisingly well with the Dark Reader (though not quite as well as fluorescein, for example). Ultimately, the only way to find out if the Dark Reader is good enough for your particular XYZ application, is give it a try.

Does the DR work with GFP?
GFP has 2 excitation peaks at 395 and 470 nm. The 470 nm peak is maximally excited by the Dark Reader and the red-shifted variants (e. g., EGFP and EYFP from ClonTech) fluoresce superbly when illuminated with DR light. In addition, the DR does not cause the rapid photobleaching of GFP that occurs with exposure to UV. Visualization of wild-type GFP is usually not practical using a Dark Reader.

Can the Dark Reader Spot Lamp be used to observe EYFP in plants?
Yes, the Dark Reader can be used to view various GFPs in both plants and animals. Here are some considerations to bear in mind:

1. The type of GFP - GFP variants such as EGFP, EYFP and dsRed work very well with the Dark Reader. Wild-type GFP does not.

2. The expression level of the GFP in the plant - There is no way of knowing if this is high enough - you simply have to try it.

3. The level of ‘background interference’. For example, chlorophyll will fluoresce and this can mask red fluorescent signals in particular. Again, you will have to try it and see.

4. The size of the samples - your sample (e.g., embryos and seedlings) may be physically too small to see any fluorescence by eye.

I want to know if the Dark Reader can be used for oligonucleotide visualization in PAGE gel on fluorescent TLC plate.
I think I understand the question - you want to separate oligos by PAGE and then put the gel on a fluorescent TLC plate and look for the ‘shadows’ caused by the oligos absorbing the excitation light. Right?
Answer is no. Oligos absorb at around 260 nm. The light from the Dark Reader is between 400-500 nm. So the oligos will not absorb the Dark Reader excitation light and the TLC plate will fluoresce behind the oligos - there will be no ‘shadow’!

How well does the Dark Reader perform with 96-well plates?
Great. Because the excitation light used by the Dark Reader passes through most plastics and glass, the Dark Reader is up to 8 times more sensitive than a 312 nm transilluminator for the detection of, for example, fluorescein in 96-well plates, centrifuge tubes, etc.

Why should I worry about UV Exposure? I use a UV box inside a hood with a CCD camera.
You are well protected, but what about your DNA samples? By the time you have adjusted the focus, fixed the zoom, and set the exposure time your DNA is well cooked. If you intend to use the DNA for further reactions you will have to contend with a significant amount of DNA damage.

Our studies show that less than 5 second exposure to UV is sufficient to significantly damage DNA. On the other hand, a 5 minute exposure on the Dark Reader resulted in no measurable damage to the DNA. (After 5 minutes on a UV box the DNA was so badly fragmented it was barely detectable.)

Clare Chemical Research

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