Drybench Plasmid.
Choosing a diagnostic restriction digest
A diagnostic digest cuts a plasmid with one or two enzymes chosen so that the band sizes on a gel tell the construct you wanted from the likely mistakes.
Correct, empty and reversed
After cloning, the common outcomes are the plasmid with the insert in the right orientation, the plasmid with no insert and the plasmid with the insert reversed. A good digest gives a different set of bands for each: for orientation, at least one enzyme must cut asymmetrically inside the insert.
The picker on this site compares all three for single and double digests of enzymes that cut the plasmid one to six times, and lists the digests whose bands differ, with the bands written out.
Reading a schematic gel
In agarose gel electrophoresis smaller linear fragments run further (Helling et al. 1974). The gel here is schematic: bands are ordered and spaced by the logarithm of size, without positions on a real gel. Fragments under 100 bp are not drawn, bands within about 5 percent of each other may run as one, and uncut plasmid runs as several forms that the drawing does not show.
See it on a real plasmid
Open the digest picker with pUC19 and an insert in its cloning site.
A digest cuts the double helix into fragments; the tour's first stop shows DNA at the scale of its bases.
References
- Helling RB, Goodman HM, Boyer HW. Analysis of endonuclease R-EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J Virol 1974;14:1235-1244. PubMed 4372397
Statements summarised by the site's builder from the references, for scientific review.