Unusual “Spiky“ bands in Gel electrophoresis from restriction digest?

Question: The attached gel image is what I had for today, with unusual spikes on the upper bands. The samples I loaded are digested plasmids (should be linear). I used 20ug of circular plasmid in a 50uL digest reaction with 20 units of the enzyme (1uL) in each. The enzyme is from NEB’s HF series, so it should be able to digest 1ug of DNA per hour; I keep the digest for 90 minutes at 37C, which would technically be enough. I also purposely overloaded the bands, so the more faint band below (linearized plasmid) is expected. However, I now expect the brighter band to be in that shape. I suspect it is the undigested plasmid, but I’ve never seen a band looking like this. If it helps, it was stained with EtBr on a 1% Agar TAE gel. Solution: In gel electrophoresis, the movement of DNA molecules, including plasmids, is influenced by their size, shape, and charge. The different conformations of a plasmid, such as supercoiled, linear, and relaxed forms, can affect their migration pattern in the gel. Supercoiled Plasmids: Supercoiled plasmids are the most compact and highly coiled form. They migrate the fastest through the gel due to their smaller size and reduced friction with the gel matrix. They can move more easily through the pores of the gel, resulting in higher mobility. Linear Plasmids: Linear plasmids are typically larger in size compared to supercoiled plasmids. They migrate slower through the gel due to increased friction and reduced mobility caused by their extended structure. Linear plasmids move through the gel as relatively slower bands. During gel electrophoresis, an electric field is applied across the gel matrix. The negatively charged DNA molecules, including plasmids, migrate toward the positive electrode (anode). The smaller and more compact the plasmid, the faster it will migrate through the gel. This movement is influenced by the resistance of the gel matrix and the size-to-charge ratio of the plasmids. #dna #gelelectrophoresis #genetics #pcr #biology