After staining and solidifying my agarose gel, I load the first well of the dried agarose gel with the TrackIt Ladder (10488058) from Invitrogen and load my unstained DNA samples into the other wells. I fill the electrophoresis device on top of the agarose gel without covering it and run the electrophoresis at 35 V (5 min) and 50 V (5 min). Then, I cover the gel with TBE 7 mm above and run the electrophoresis at 65 V (60 min).
When I take the photo with Trans UV BioRad, the ladder bands are distinct and stain well, but the DNA samples are not.
I need help with this, I have tried it with other Invitrogen ladders and without them with the same procedure and the DNA bands are visible, except on this TrackIt Invitrogen ladder.
you do appear tp have weakly visible bands in this image so I would check that your sample loading dye is dense enough to hold the sample in the well. If so then run 3 or 4 more cycles of pct to get more product. You mention that the gel is not covered with running buffer while loading. It is usual to cover the gel completely with running buffer then load the samples through the buffer with a dense loading dye in sucrose or glycerol to get good results. Loading sa,ples then covering the gel with buffer may disturb the samples in the wells and lead to some sample lossYou could just load more sample and the gel would look better in this case but more cycles would be better
Try doing a PCR run and then reanalyse the bands.I guess you’ll get more defined bands.
Hello again, I can finally solve it!! It was the dilution factor of my ladder, because it was very concentrated (despite being prepared according to the manufacturer's instructions), and the camera focused only on the brightness of the ladder. After several tests, I found the correct ladder's dilution, and the electrophoresis has a good profile. I didn't change anything in the PCR
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