Problems for DNA agarose gel visualization with a gel documentation system - (Feb/05/2019 )
When performing bacterial strain typing we got a problem for DNA agarose gel visualization. After performing bacterial DNA extraction, the extracts were run in an electrophoresis equipment using 1% agarose gels in TBE buffer with ethidium bromide from Sigma-Aldrich as fluorescent mark. We loaded a gel well with 4µL of a mix of 6X DNA loading dye Thermo Scientific (2 µL) with the DNA sample (2 µL). In another well we used 3.5 µL of a mix of 6X DNA loading dye (2 µL ) with DNA ladder GeneRuler 1 kb from Thermo scientific (1.5 µL). The electrophoresis was carried out at 100 V until the dye reached 2/3 of the gel, during approximately 40 min.
After that, we put the gels in the chamber of a BIOTOP Fluor Shot - gel documentation system, purchased from a Chinese biotech brand Shangai Biotech Co. The UV transilluminance lamp mode was used (312 nm, according to the manufacturer). We could not see any band in the images displayed by the equipment software. We performed the same procedure different times, and also tried incorporating EtBr to the buffer bath instead, but again we failed to visualize any bands in the gels. We also tried to run some samples of DNA enzyme digests using 2% agarose gels, but the results were the same. Initially we thought that perhaps the lamp of the equipment was not working, but we opened the door using the “gel cut” mode and could see that the transilluminator lamp was on, with the bottom screen being evenly illuminated with violet light.
Ragarding the gel documentation equipment: we purchased the equipment about 2 years ago. Unfortunately we could not test it until recently since we were just acquiring the complete set of apparatus for molecular biology assays. I tried to contact the Chinese company customer service but they ignored my query. Please advice as to what could be causing the problems for visualizing the DNA agarose gels.
What I would do first of all is get a known amount of DNA and do some serial dilutions and run on a gel. Pre-made ladders/molecular mass markers are good for this, as they usually come with known concentrations, so you can work out detection limits etc. Make sure you have optimal running conditions for the gel, and stain in EtBr after running. Ensure that the gel sits on the transilluminator directly (i.e. not in the gel tray) when visualizing. If possible have a look at the gel on another system to check that you can see everything as expected, then take it to your "new" one.
Have a look at the gels in "cut mode" and see if you can see bands on the gel itself. If you can, it's not a problem with the transilluminator itself, but likely an imaging/camera/settings issue. If you can't, there are a couple of possibilities:
1) the violet light you have seen is not what you think it is - perhaps it is a blue-light transilluminator, which don't cause fluorescence in EtBr, or the color is caused by a filter on the bulbs or between the lamp and the gel (e.g. white light buls with colored filter) - and is not UV at all - in which case you have a fraudulent sale on your hands.... You can test these sorts of thing with fluorescent substances like chalk as well as using the gel.
2) Something is blocking the UV - check for plastic layers on the transilluminator which could be attenuating the UV or anything that could be blocking the light at all.
3) The light is too weak - the power output just isn't enough to illuminate. Transilluminators will really light up a room when switched on.
To the excellent suggestions above I would add that you can buy plastic rulers or test sheets that are printed with an ink that looks orange in normal light and fluoresces like EtBr under UV; you can use that for troubleshooting instead of wasting a lot of gels/DNA samples.They should not be expensive. Some companies even give these away at product shows, or include them in the transilluminator kit when you buy your equipment. They help you get the correct focus, too, since you can judge the clarity of the words & numbers in the image. That ought to tell you whether the problem is the gels/samples or the detection system.
Make sure your system is designed for detection of EtBr and not Sybr green or some other dye. There should be a filter between the the illuminated gel and the "camera" (detector) and it will be dye-specific. For EtBr it will appear orange-yellow. Maybe the filter has not been installed? Sometimes you have to buy these separately. The imager I used had a rotating holder for 4 different filters and you had to make sure you had rotated to the correct one, because it always defaulted to the wrong one. Are you getting a blank white image or just black background?
Don't forget to protect your eyes when looking at the light source!
bob1 on Wed Feb 6 04:41:50 2019 said:
What I would do first of all is get a known amount of DNA and do some serial dilutions and run on a gel. Pre-made ladders/molecular mass markers are good for this, as they usually come with known concentrations, so you can work out detection limits etc. Make sure you have optimal running conditions for the gel, and stain in EtBr after running. Ensure that the gel sits on the transilluminator directly (i.e. not in the gel tray) when visualizing. If possible have a look at the gel on another system to check that you can see everything as expected, then take it to your "new" one.
Have a look at the gels in "cut mode" and see if you can see bands on the gel itself. If you can, it's not a problem with the transilluminator itself, but likely an imaging/camera/settings issue. If you can't, there are a couple of possibilities:
1) the violet light you have seen is not what you think it is - perhaps it is a blue-light transilluminator, which don't cause fluorescence in EtBr, or the color is caused by a filter on the bulbs or between the lamp and the gel (e.g. white light buls with colored filter) - and is not UV at all - in which case you have a fraudulent sale on your hands.... You can test these sorts of thing with fluorescent substances like chalk as well as using the gel.
2) Something is blocking the UV - check for plastic layers on the transilluminator which could be attenuating the UV or anything that could be blocking the light at all.
3) The light is too weak - the power output just isn't enough to illuminate. Transilluminators will really light up a room when switched on.
Bob1:
Many thanks for your thorough and useful answer to my post. I have run the DNA ladder several times, but hadn´t gotten any image, so I think I should try another system to see if I can see the fluorescent bands. Do you now if an equipment like the following would be suitable?
I asked the manufacturer, but they didn´t reply to me either.
As for the chalk for testing the equipment, which type of chalk you recommend?, is there a commercial brand?
Thanks again and regards.
OldCloner on Wed Feb 6 14:18:56 2019 said:
To the excellent suggestions above I would add that you can buy plastic rulers or test sheets that are printed with an ink that looks orange in normal light and fluoresces like EtBr under UV; you can use that for troubleshooting instead of wasting a lot of gels/DNA samples.They should not be expensive. Some companies even give these away at product shows, or include them in the transilluminator kit when you buy your equipment. They help you get the correct focus, too, since you can judge the clarity of the words & numbers in the image. That ought to tell you whether the problem is the gels/samples or the detection system.
Make sure your system is designed for detection of EtBr and not Sybr green or some other dye. There should be a filter between the the illuminated gel and the "camera" (detector) and it will be dye-specific. For EtBr it will appear orange-yellow. Maybe the filter has not been installed? Sometimes you have to buy these separately. The imager I used had a rotating holder for 4 different filters and you had to make sure you had rotated to the correct one, because it always defaulted to the wrong one. Are you getting a blank white image or just black background?
Don't forget to protect your eyes when looking at the light source!
Hello OldCloner:
Thank you very much for your valuable comments. I have additional comments/questions regarding your post.
1. Do you know if the plastic rulers that you mentioned are commercially available? It seems as a very practical way to test the equipment. I am afraid that if I just use a compound such as a powder with fluorescence at ~310 nm I would not see any images as the product senses the transilluminance of the samples.
2. I checked the lenses of the imager and it seems that it has an orange filter as you implied. It seems it only has one optical mode, and two illumination modes (reflected white and "UV transilluminator").
3. In the images we just get a black background. At some point we could barely see a very dim band for the marker as we doubled the amount of EtBr, but when we repeated the run we couldn´t see any band. This may indicate that the illumination is not good...
Best regards.
rogevala: That system should be fine - it is to check that you don't have something wrong with your gel before you test it on your new system.
The systems that OldCloner mentioned are a better option for testing than chalk, which would be messy. They look like a ruler or square with both black markings and fluorescent markings (lines, dots, grids etc) that can be used to test the imaging sensitivity and focus. Biorad has this ruler available. However, literally anything that fluoresces under "blacklight" can be used for this, so even a sheet of black paper with some fluorescent highlighter would probably work. Bic (company) produces a good range of fluorescent highlighters that are available world-wide.
bob1 on Wed Feb 6 21:22:01 2019 said:
rogevala: That system should be fine - it is to check that you don't have something wrong with your gel before you test it on your new system.
The systems that OldCloner mentioned are a better option for testing than chalk, which would be messy. They look like a ruler or square with both black markings and fluorescent markings (lines, dots, grids etc) that can be used to test the imaging sensitivity and focus. Biorad has this ruler available. However, literally anything that fluoresces under "blacklight" can be used for this, so even a sheet of black paper with some fluorescent highlighter would probably work. Bic (company) produces a good range of fluorescent highlighters that are available world-wide.
Thanks for your answer. I will try all this. Regards.
OK, the black background means you have the filter in place. If you were getting a “whiteout” that usually means the filter is missing- or gross overexposure. Can you control exposure times? Do you have an F-stop (controls aperture size on a camera) or digital equivalent? If you saw a dim band, you might just need longer exposures or a more open aperture (lower number f-stop). For white light photography you would need a higher F-stop and shorter exposure times, and the system might default to that. In the system I had both of these settings had to be done manually on the camera lens. Fortunately, being an Old Cloner, I had experience with manual cameras from the pre-digital era.
When you opened the door in “gel cut mode” was there a gel in there? Could you see the bands then?
Bob1 answered the Q about the ruler. I think I had that BioRad ruler, but I got it as a freebie at an ASM meeting long ago!
A 310 wavelength is not the best for exciting EtBr, but it probably induces less damage to the DNA in the gel in case you want to recover it for cloning. Also there would be less photo-bleaching of the stain in the gel (meaning if you let the gel sit a long time on the UV light at shorter wavelengths, the stain fades away quickly.) Older units often had U.V. settings in the 200's and gave very bright images, but if you let the gel sit there a few minutes, it would fade away and had to be re-stained.
OldCloner on Thu Feb 7 18:06:29 2019 said:
OK, the black background means you have the filter in place. If you were getting a “whiteout” that usually means the filter is missing- or gross overexposure. Can you control exposure times? Do you have an F-stop (controls aperture size on a camera) or digital equivalent? If you saw a dim band, you might just need longer exposures or a more open aperture (lower number f-stop). For white light photography you would need a higher F-stop and shorter exposure times, and the system might default to that. In the system I had both of these settings had to be done manually on the camera lens. Fortunately, being an Old Cloner, I had experience with manual cameras from the pre-digital era.
When you opened the door in “gel cut mode” was there a gel in there? Could you see the bands then?
Bob1 answered the Q about the ruler. I think I had that BioRad ruler, but I got it as a freebie at an ASM meeting long ago!
A 310 wavelength is not the best for exciting EtBr, but it probably induces less damage to the DNA in the gel in case you want to recover it for cloning. Also there would be less photo-bleaching of the stain in the gel (meaning if you let the gel sit a long time on the UV light at shorter wavelengths, the stain fades away quickly.) Older units often had U.V. settings in the 200's and gave very bright images, but if you let the gel sit there a few minutes, it would fade away and had to be re-stained.
Many thanks OldCloner, I did try to adjust the optical parameters of the imager, but it did not work. I will try the ruler to test the equipment and see if I can use an additional UV transilluminator to see if my DNA samples are alright. I may also try another DNA ladder that I have (100 kbp). Regards!
Hello Bob1 and OldCloner;
I just want to give you a quick update on my gel visualization problem. We finally managed to see the bands. The advices that I received from you were very useful to solve the problem. Apparently the problem with my gels were that we were overheating the EtBr during the preparation of the gel. Other additional issue was that the TBE buffer was too concentrated. Now we can see the DNA ruler very clearly. Here I attach a picture of it.
Now we are struggling a bit with the electrophoresis after PCR. Our sample bands fall in a very low molecular weight range (below the lowest limit) and we were supposed to have a band approximately at 1.5 kb. But we are trying to improve our amplification technique. This problem can be observed in the attached picture.
I thank you very much for your help. Regards.