Why the electrophoresis speed is different for the three morphologies in the DNA plasmid

Normal plasmids are closed double-stranded DNA. During electrophoresis, the plasmid may be loop-opened, or the loop-opening spiral may become linear. These three forms have different separation rates and different separation speeds during electrophoresis, and are divided into three bands.

The helix of closed loop (supercoiled), open loop, and linear shape decreases sequentially. (Ring-opening plasmid refers to the double-stranded circular plasmid DNA that is partially dissolved, so the electrophoresis speed is the slower) The order of the three conformational plasmids in agarose electrophoresis is supercoiled "linear" ring-open, the linear plasmid is in the middle, and the ring-opening plasmid is at the end.

One indicator of how good a plasmid is is the amount of the supercoiled plasmid in all the plasmids.

Plasmid DNA has three different configurations: OC configuration, L configuration, and SC configuration. At the front of electrophoresis is the SC configuration.

Characteristics of plasmid DNA electrophoresis: Number of DNA chromogenic bands: There are 3 bands in plasmid DNA electrophoresis; Genomic DNA should have only one or two bands. Because:

Genomic DNA electrophoresis is generally 1 band, although it can also break during your extraction, forming large fragments of tens to hundreds of kb. But we generally use 1% glue, which can't distinguish between different sizes of DNA, so it looks like a band. If the prepared glue is added with Lamda Hindiii marker, and the glue running time is appropriately extended, there should be several bands.

Meaning:

DNA purity, buffers.

Temperature conditions and restriction enzymes.

itself affects the activity of restriction enzymes. Most restriction enzymes are not affected by RNA or single-stranded DNA. When trace amounts of contaminants enter the restriction enzyme stock, it can affect its further use, so use a new pipette tip each time when aspirating the restriction enzyme.

If two restriction enzymes are used, care must be taken to provide the optimal salt concentration for each.

Gel electrophoresis separates DNA of different lengths due to the interaction of molecular sieves and charge.

First, there are pores inside the gel from which the DNA is isolated, forming a sieve-like pore size. The higher the molecular weight of the DNA, the slower it will swim in the gel, and the smaller the molecular weight of the DNA, the faster it will swim in the gel. Different lengths of DNA mean different molecular weights and therefore different surge locations.

Second, the DNA molecule is negatively charged in a pH solution above the isoelectric point, moving towards the positive electrode in the electric field. Due to the structurally repetitive nature of the sugar-phosphate backbone, the same amount of double-stranded DNA has almost the same amount of net charge, so they can move towards the positive electrode at the same rate.

The molecular sieve effect and the charge effect can lead to the separation of different sizes of DNA at different electrophoresis positions