A student runs an uncut plasmid of 3800 bp on a gel…
Contributed by StemPy LabTry it on paper first. A hint, the key idea and the worked solution are here when you need them.
A student runs an uncut plasmid of bp on a gel. She sees two bands: one that runs like bp and one that runs like bp. She then cuts the plasmid with an enzyme that has one site, and sees one band.
- Which band of the uncut plasmid is the supercoiled form, and why does it run so far?
- What is the other band?
- At what size does the single band of the digest run, and why?
Need a reminder? The key ideaShow
Read a size from a ladder with a straight line of size against distance. An uncut plasmid shows a supercoiled band (fastest), a linear band and a nicked band (slowest). The control plates are: no DNA, uncut vector, cut vector without ligase, and cut vector with ligase and no insert. Efficiency colonies (ng of DNA on the plate) for each microgram.
The worked solution
Have you tried it?
Work it out on paper first. It is fine to get stuck or make a mistake: that is how we learn. When you are ready, go through the solution one step at a time, and check each step against your own.
Given. Uncut plasmid with bands that run like and bp. A digest with one band.
Plan. Link each band to a shape of the plasmid.
Step 1. (a) The band that runs like bp is the supercoiled form. It is tightly twisted and compact, so it passes through the gel more easily and runs farther.
Step 2. (b) The band that runs like bp is the nicked (open circular) form. It is loose and bulky, so it runs slowly.
Step 3. (c) The enzyme cuts the circle once, so a line of bp forms. It runs at its true size, bp, between the other two.
Answer: (a) The bp band, because it is compact. (b) The nicked form. (c) At bp, because a plasmid cut once is a linear molecule.
Watch out. The supercoiled band is not a smaller plasmid. It is the same DNA in another shape.
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