Hi,
I am trying to visualize my DNA bands run on agarose gels (stained with gel red during the gel preparation). Although I am using UV fluorescence to visualize the gel, I think some setting in my UV machine has been changed because I always obtain images like those attached. The bands of the marker are too thick and bright that I can't distinguish them. On what can this effect depend? What parameter could be involved? I am sure that it is not a problem with the amount of the marker loaded because all my gels are like this.Thanks
I have found that decreasing the amount of Gel Red used, especially for the ladder, helps get clearer (and straighter) bands. If you are adding the Gel Red to the gel, try instead mixing it in the loading dye instead: 1 uL Gel red in 1 mL loading dye (mix well).
Also, I think zooming in so that your gel fills up the whole screen will help, as will inverting the images so that you see black bands on a grey background instead of white bands (see attached image. Excuse the primer dimers, this was during optimization). That also uses less ink during printing. The manual for the imager will give tips on how to adjust the image, but play around with the brightness and shading functions until you get an image you are happy with.
You say that this happens with every photo you take. Instead of looking at the gel or the light box, could it be something as simple as the camera losing the filter for the light emitted from the gel? Admittedly on blue light box transilluminators, the camera will still give a picture of the gel, but the contrast is all wrong if the wee orange filter that sits up inside the camera hood has dropped off, and I vaguely remember something similar happening with the UV box 20 years ago (with the old Polaroid Instant cameras!).
I have experimented with Gel Red and found it useful in checking for the presence of a PCR product, and much less useful when looking at PCR banding patterns.
The stain is bound to a large molecule to stop it getting absorbed by human cells but the PCR products will not run crisp and clear through the agarose now. The ladder bands will run into each other.This is problematic and I found solution but move on from GelRed.
I would advise using SYBR-Safe as an ErBr replacement - I have got much better results with it - nice clean and crisp bands and similarly for ladder bands.
Hope this helps,
Robert.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology