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I haven't seen any gel lanes in neither my PCR samples nor the marker itself when photographing them. This is not my first time performing 1.2% agarose gel electrophoresis.

I see gel lanes when I take the gel out the apparatus, but when I go to photograph them: nothing shows up. I only let the samples "run" for 30-40 minutes (when I ran for an hour, the bands almost ran off the gel).

Tried using different markers: no luck.

Re-added some more EtBr to better stain the gel: no luck.

I am also keeping the lights off in the dark room, so I do not think I am exposing the film to light early on (errors still a possibility though).

What could I be doing wrong? Am I taking the film out too early?

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I don't understand the part of your question where you refer to film in a darkroom. If you are staining with EtBr then you must be using a UV lamp to visualise the bands. There is usually no question of "taking film out" since the gel is usually photographed with a digital system of some kind. Please explain exactly what you do with the stained gel. –  Alan Boyd Mar 7 at 6:11
    
Do you see the color from the loading dye migrating through the gel? –  Chris Mar 7 at 7:34
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2 Answers 2

What I can quickly think of is that you were running the gel incorrectly: instead of from - to + direction, You ran it the other way around and your DNA went out of the gel. Another thing might be that the UV light was not turned on or is broken.

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Here's my $0.02 at troubleshooting:

I see gel lanes when I take the gel out the apparatus,

Good, you're loading your gel correctly and the density is correct so the DNA sits at the bottom of the wells.

but when I go to photograph them: nothing shows up.

This points to a problem with image formation: not enough EtBr, not enough DNA, burnt out UV bulb, incorrect UV filter, or incorrect imaging station settings.

I only let the samples "run" for 30-40 minutes (when I ran for an hour, the bands almost ran off the gel).

You've run the electrophoresis in the correct direction and not long enough to run the bands completely out of the gel.

Tried using different markers: no luck.

Are you loading enough DNA with these markers?

Re-added some more EtBr to better stain the gel: no luck.

I suspect a problem with the gel documentation system.

I am also keeping the lights off in the dark room, so I do not think I am exposing the film to light early on (errors still a possibility though). Am I taking the film out too early?

I don't know what you're talking about here. EtBr is a fluorescent dye under UV illumination, and doesn't normally require photographic film. Some systems will capture an image of the gel using a "Polaroid" sort of camera, but that works because the gel was exposed to UV light and the incoming light was filtered accordingly. Most systems however use a gel documentation system with a transilluminating tray and digital camera system to take an image.

Some things to try:

1) Prepare two samples: a standard marker lane, and a DNA lane of known concentration (load up a lot of DNA, like 1-2 ug of plasmid to know that you can't miss the band). Add 1 uL stock EtBr to each lane's sample, mix, then load on the gel. Run the gel using your usual parameters, keeping a eye on the migrating dye front the make sure you don't run the DNA off the gel. This will make sure that you have two good samples that should appear on the gel, and that they are plenty bright by having excess EtBr.

2) Image the gel. Things to double check here:

  • is the emission wavelength correct? EtBr requires short or medium wave UV illumination (250-300 nm), but some systems have long wave illumination as well. Long wave will not work.

  • is there filter setting correct? EtBr fluoresces with a red peak (595 nm max), and if a filter is in place to remove this band, you won't see anything either. Usually there is no filter, or an "EtBr/UV" filter option. Here's a technical document with an example of setting up for EtBr capture.

  • is the camera set with an appropriate shutter speed/exposure time? Try experimenting with 20 ms to 200 ms. On our system, 40 ms is ideal. Too short an exposure time means that you won't see anything.

  • is the camera iris set the maximally open? If the iris is closed too much, it restricts the incoming light for image formation and eiter no image or a weak image will form. This will also impact ideal exposure time (see above).

  • is the camera in focus and operational? Can you image an object like your gloved hand or the physical gel before getting the EtBr settings correct?

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