Hi, I noticed something weird during agarose gel electrophoresis. I use SYBR Green to stain the DNA. When my DNA sample shows faint bands, the ladder also looks faint. But as far as I know the ladder shouldn’t be affected by the DNA quality. On days when the DNA is good, the ladder looks bright too along with the bands. Is there a reason why both the DNA and the ladder show up faint or bright together?
" possibilities are that 1 the bulbs on the transilluminator are failing and when you align your gel over a good bulb then everything is strongly fluorescent but over a weak bulb everything is faint. Possibly also someone else is changing the transilluminator filter/settings.. Move the gel around and take pictures or check that all bulbs are lit up
and 2 your pipettes need recalibrating or new seals and addition of the dye to the samples is variable so with one pipette everything gets too little dye when using a leaky pipette. Try weighing 10x 20ul of water to check that your pipette is delivering the right volumes.
When both your DNA samples and the ladder appear faint, it is not actually due to the DNA quality itself. Instead, it’s more likely related to the gel staining process,
If the TAE/TBE buffer is old, incorrectly diluted, or reused many times, it can reduce DNA migration efficiency, affect SYBR Green fluorescence due to pH changes or ion depletion.
Use fresh 1X TAE/TBE buffer. Make sure your gel and running buffer are both made with the same batch
The issue is with either the stain or the imaging.
SYBR Green is sensitive to light & to freeze-thaw cycles. It loses brightness as it gets degraded.
Get a fresh aliquot from the freezer, make sure the camera & light box are adjusted the way you want, and I bet you will get much better images.
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