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Clare Chemical Research
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Frequently Asked Questions
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| 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 orange glasses for eye protection or other reasons? I want to double check the actual size of the blue light area vs the outside diameter of the entire box. How resistant is the blue screen to scratching? Which has higher intensity per unit area, the DR45 or DR88, so I can detect less protein stained with SYPRO? What about solarization of the blue screen? How long do the lamps last? FAQ - Imaging I would like to know if the DR works well with Polaroid cameras. I would like to know if the DR45 transilluminator would work with our XYZ imager. We have a camera set up for EtBr gel photography using a UV transilluminator. Do you think we would need a new filter to take images using a Dark Reader? We use your Dark Reader with SYBR Gold for DGGE gels. We use a Polaroid camera following exactly the procedure on your web site. We use the amber screen to apply onto the gel, but our pictures are more fuzzy than those taken on our UV box. I would like to buy a Dark Reader camera filter for my XYZ camera. Do you have an idea what filter fits? I do not see a 58 mm DR filter listed on your web site. I need information and price about the appropriate filter. Do you recommed to use a filter onto the camera of a geldoc or to use directly amber screen? When I image a gel using my CCD camera, I get a very high background that almost looks like a flare. How can I fix this? I am using fluorescein and rhodamine and I want to know how to separate the green and red channel information in my color image. 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 I would like to know if I can leave it placed on top of a glass plate whilst on top of the dark reader permanently to both view & capture an image via a digital camera? Using EtBr on a UV transilluminator gave more concise DNA bands with less smearing. On the otherhand, the SYBR Green + Dark Reader results had very high levels of background smearing extending from the loading margin all the way down to the product. Using SYBR Green with your Dark Reader, rather than EtBr and a UV unit, I found that there was no clear difference in band intensity across the different concentrations which prevented DNA quantification. On your web page you claim that it is possible to see about 5 ng of DNA stained with EtBr by eye. In our hands (or rather: to our eyes), bands below 50 - 100 ng are very hard to see by eye. What could be wrong? Light bulbs? Orange screen? FAQ - More Why `Dark Reader`? I would like to know if the fluorophor XYZ is compatible with the Dark Reader. Does the DR work with Green Fluorescent Protein? Can the Dark Reader lamps be used to observe EYFP in plants? I want to know if the Dark Reader can 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. |
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| FAQ - Dark Reader Components Do we have to use the orange glasses for eye protection or other reasons? 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. I want to double check the actual size of the blue light area vs the outside diameter of the entire box. The viewing surface is 21 x 14 cm. The outer dimensions are only somewhat larger at 25 x 18 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 does not appear to be 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 $70-100 whereas a new UV screen will cost about $500. 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 per unit area, the DR45 or DR88, so I can detect less protein stained with SYPRO? The DR45 outputs a little more light per unit area than the DR88 but this is not necessarily going to help you detect smaller amounts of SYPRO. The detection level is determined by the fluorescence signal-to-background ratio and this will be about the same for both units. (There are some situations where having more light can help, but I don’t think this is one of them - you are going to be limited by the background fluorescence from the gel itself.) What about solarization of the blue screen? The screens used in UV boxes become solarized very quickly - after 48 hours of continuous use the UV light intensity drops by over 50%. In contrast, a DR45 transilluminator in heavy use in the CCR lab for over 4 years (about 1000 hrs of continuous use) showed a drop in light intensity of only 25%. How long do the lamps last? The lamps used in the DR-45 and DR88 transilluminators are rated for 10,000 hours (about 13 months of 24 hours-a-day operation) and the DR-195 lamps for 6,000 hours. Replacement lamps cost about $15 and $30 respectively. In contrast, typical UV bulbs are rated at 5000 hours and cost $40 each. 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 if the DR works well with Polaroid cameras. If you mean the old DS34 film camera, yes it does. If you are using the camera attached to a hood, then it is better to use a DR camera filter - placing the hood on the amber screen raises the camera about 1/4” and, at an f-stop of 4.5, the narrow depth of field will result in a ‘fuzzy’ photo. You can always go to an f-stop of 5.6, but you will need to increase the exposure time. As to the newer Polaroid digital camera - the ‘spec. sheet’ does not look very impressive at all. I would like to know if the DR45 transilluminator would 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 issue: - the physical size of the cabinet - is it big enough for the DR45. - the electrical power supply into the cabinet. The XYZ may use a different style plug so you may need an adapter (a Radioshack part). - the CCD camera may not have a built-in IR filter, so you will need to get a separate one. We have a camera set up for EtBr gel photography using a UV transilluminator. Do you think we would need a new filter to take images using a Dark Reader? 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. We use your Dark Reader with SYBR Gold for DGGE gels. We use a Polaroid camera (No.667, 665 film) following exactly the procedure on your web site. We use the amber screen to apply onto the gel, but our pictures are more fuzzy than those taken on our UV box. 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. - 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. Do you have an idea what filter fits? This question should be addressed to whoever supplied the camera. I do not see a 58 mm DR filter listed on your web site. I need information and price about the appropriate filter. We do not currently have a 58 mm filter. but you can always just use the amber screen that comes with the transilluminator. It works exactly like a DR camera filter. However, you may decide you must have a camera filter for a couple of reasons: - if your fluorescent samples are on the bulky side - over 6 mm thick - they will not fit under the amber screen. - condensation can form on the underside of the amber screen, especially when photographing warm gels, which obscures the fluorescence. If attaching a filter to the camera is indeed essential then order a 62mm Dark Reader filter and a 58-to-62 mm step-up ring. The 58-end of the step-up ring screws to the mount and the 62-end holds the filter. Step-up rings are readily available from photogaphic stores. Do you recommed to use a filter onto the camera of a geldoc or to use directly 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. See above answer. When I image a gel using my CCD camera, I get a very high background that almost looks like a flare. How can I fix this? This sounds like IR radiation. All lamps emit IR radiation. Unfortunately, this is the region of the spectrum to which CCD chips are most sensitive. (Oddly enough, the more expensive the CCD camera, the less likely it seems it will have a built-in IR-blocking filter, but all the basic cameras from the likes of Olympus, Kodak and Nikon have a built-in IR filter) The lack of an IR filter will result in excessive background that effectively obscures any fluorescence signal. The solution is to attach an IR-blocking filter to the camera. These are available from a variety of sources. Tiffen sell several different sizes for around $50. I am using fluorescein and rhodamine and I want to know how to separate the green and red channel information in my color image. Image manipulation techniques, such as multi-color analysis, background subtraction and excitation intensity normalization are beyond the scope of these FAQ. Briefly, the software we use for image analysis is IGOR Pro from Wavemetrics which has a number of built-in image manipulation routines and can also perform calculations on images. 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 5 ng. On Polaroid film, for SYBR Gold, SYBR Green and GelStar, the detection is 25 - 50 pg. The ethidium detection limit on Polaroid film is around 650 pg using our AF-09 camera filter - about twice as good as without the filter. With a CCD camera system it is possible to detect 10 - 20 pg of SYBR- or GelStar-stained dsDNA. Ethidium detectability is about the same as with Polaroid film. The new SYBR dye - SYBR Safe - is less sensitive than Green and 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 SYBR Gold and SYBR Green 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 DNA molecular weight 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 I would like to know if I can leave it placed on top of a glass plate whilst on top of the dark reader permanently to both view & capture an image via a digital camera? 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. Using EtBr on a UV transilluminator gave more concise DNA bands with less smearing. On the otherhand, the SYBR Green + Dark Reader results had very high levels of background smearing extending from the loading margin all the way down to the product. Too much DNA! The SYBR + Dark Reader combo is at least 5 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. Using SYBR Green with your Dark Reader, rather than EtBr and a UV unit, I found that there was no clear difference in band intensity across the different concentrations which prevented DNA quantification. Too much DNA! The increased sensitivity of the SYBR + Dark Reader combination means that your DNA fluorescence intensities are saturated. To get back in the linear range, load 5 - 10 times less DNA on the gel. On your web page you claim that it is possible to see about 5 ng of DNA stained with EtBr by eye. In our hands (or rather: to our eyes), bands below 50 - 100 ng are very hard to see by eye. What could be wrong? Light bulbs? Orange screen? The sensitivity of the Dark Reader with EtBr is always going to be lower than using a UV box. This is because EtBr is better excited in the UV than in the visible. To see the 5 ng of DNA you have to be in a very well darkened room and let your eyes adjust to the faint fluorescence. Factors affecting the sensitivity level include the amount of EtBr that you stain the gel with. For example, we use 0.1-0.2 ug/mL of EtBr and this helps reduce the background signal. Also, most of the gels we run are mini-gels with small wells. If you are using bigger wells, then the DNA bands will be wider and so the DNA will be more spread out. If you need more sensitivity then you should take a look at the new SYBR dyes from Molecular Probes. These work really well with the Dark Reader. In fact, you will need to cut down the amount of DNA you normally load on the gel - otherwise you will start to see all sorts of minor bands that you never knew you had! FAQ - More Why `Dark Reader`? Very simple: switch on a UV box in a darkened room and the walls (not to mention your teeth!) glow purple. Switch on the Dark Reader and the room remains just that - dark. It is important to remember that is not the amount of light but the quality of light that is important when looking at fluorescent DNA bands in a gel. With a UV box you are looking at bright DNA bands against a blazing UV background (that is not doing you or your DNA any good!). With the Dark Reader, give your eyes a few seconds to adjust and the fluorescent DNA bands stand out just as well, if not better, than on a UV box. 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 Green Fluorescent Protein? 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 lamps 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.) |
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Clare Chemical Research
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