I ran a PCR product of ~300 bp on a 2% TAE-agarose gel for 30 minutes. I used Sy
ID: 31829 • Letter: I
Question
I ran a PCR product of ~300 bp on a 2% TAE-agarose gel for 30 minutes. I used Sybr-safe as a DNA stain. Voltage was 80V.
When I imaged the gel, the DNA on the bottom half of the gel, including the ladder had disappeared (it showed no bands). The DNA in the top half looked-clear and well-separated, but the bands on the bottom were somehow missing.
What could be the reason? I've never encountered this before.
I know it's not due to gel overheating - I ran the gel in the cold room and when it was done it was cold to the touch.
Explanation / Answer
If you use pre-staining, the stain migrates as well in the electric field and this can lead to some parts of the gel being unstained. This would look pretty much like you describe, the ladder being invisible as well points strongly to that part of the gel just being no stained properly.
It sounds a bit unlikely with your running parameters, I've only seen that with a very long running gel, but I also haven't used pre-staining for quite a while now. Ethidium bromide is positively charged and migrates in the opposite direction as the DNA, I have no experience with Sybr-Safe though and don't know if it behaves the same as EtBr in this regard.