FAQ - Dark Reader Components
The glasses are not for eye protection. (Indeed, do not use the glasses with a UV unit because they do NOT provide protection against UV !)
The light from the Dark Reader is 100% visible blue. It is 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 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. The glasses 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.
The viewing surface is 15 x 19 cm. The outer dimensions are only somewhat larger at 18.5 x 22.5 cm. Check out the transilluminator page for more physical dimensions on all our units.
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 $200 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.)
The DR46 and DR89 (indeed, all our transilluminators) have almost identical light intensities. Note that 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.
The LEDs used in all our units are rated for 50,000 hours (over 17 years of 8 hours-a-day operation!).
FAQ - Imaging
Just about any digital camera can be used – your cell phone can take a great photo of a DNA gel. In general though, a digital camera with manual control of exposure time, focus and f-stop is preferred. Many researchers like the Canon G-series cameras.
Yes it will:
There are a couple of possible issues:
– the physical size of the cabinet – is it big enough for the Dark Reader Transilluminator? * check the physical dimensions here*
– the electrical power supply into the cabinet. Your imager may use a different style plug so you may need an adapter. We have several styles available.
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.
There are a couple of possibilities:
– 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.
-Your camera is in ‘auto’ mode.The extraordinary lighting conditions involved in photographingfluorescent DNA gels may confuse auto-focus. Switch to ‘manual’ mode.
This question should be addressed to whoever supplied the camera. The specifications for lots of cameras can be found on www.dpreview.com
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
Summary of the most common stains:
| Viridi Vivid, SYBR Gold | < 100pg |
|---|---|
| SYBR Green , GelStar | ~ 200 pg |
| GelGreen | ~ 300pg |
| SYBR Safe | < 1ng |
| EtBr | > 5ng |
The purchase price of the new DNA stains is definitely 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 $2 if stained with EtBr and $1.50 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.
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.
Too much DNA! Many of the new stains are at least 10 times as sensitive as EtBr. 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.
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
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.)
The excitation maximum may seem to be a bit on the long side. However, from looking at the entire spectrum there is significant excitation below 500 nm and this is what the Dark Reader would ‘go after’. Tetramethyl rhodamine (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). If you are uncertain about your particular application, please send us an email and we can advise on the suitability or even arrange for a demo unit for you to try!
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) 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.
Yes, the Dark Reader can be used to easily and safely view Fluorescent Proteins in plants, animals and micro-organisms. Below are a few considerations to bear in mind.
And remember: you can always contact us to discuss your particular experimental set-up at tech_support.
1. The type of Fluorescent Protein – variants such as EGFP, EYFP, dsRed and mCherry work very well with the Dark Reader. Wild-type GFP does not.
2. The level of ‘background interference’. For example, chlorophyll will fluoresce and this can mask red fluorescent signals in particular.
3. The expression level of the GFP in the organism – It is difficult to know in advance if this is high enough – you will probably have to try a Lamp and see. We do usually
have demo Lamps available. Contact us at demos .
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 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?
Unfortunately, the 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’!
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 UV transilluminator for the detection of, for example, fluorescein in 96-well plates, centrifuge tubes, etc.
