What are the different conformations of plasmid DNA?
Plasmid DNA can exist in three conformations: supercoiled, open-circular (oc), and linear (supercoiled plasmid DNA is often referred to as covalently closed circular DNA, ccc).
How does gel concentration affect gel electrophoresis?
The concentration of gel affects the resolution of DNA separation. The agarose gel is composed of microscopic pores through which the molecules travel, and there is an inverse relationship between the pore size of the agarose gel and the concentration – pore size decreases as the density of agarose fibers increases.
How many bands should appear on the agarose gel when your plasmid is uncut by restriction enzymes?
3 bands
When uncut plasmid DNA is isolated and run on an agarose gel, you are likely to see 3 bands. This is due to the fact that the circular DNA takes on several conformations the most abundant being: supercoiled, relaxed and nicked.
What determines the concentration of agarose you add to your gel?
Agarose gels are prepared using a w/v percentage solution. The concentration of agarose in a gel will depend on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-2%. The volume of the buffer should not be greater than 1/3 of the capacity of the flask.
Are plasmids linear?
Plasmids are usually circular molecules of DNA, although occasionally, plasmids that are linear or made of RNA exist. They may be found as single or multiple copies and may carry from half a dozen to several hundred genes. Plasmids can only multiply inside a host cell.
Why do you need to cleave plasmids before running them on a gel?
To cut DNA, RNA, or plasmid at restriction sites (like EcoRI, BamHI, hindIII and BglII) to create smaller genetic fragments that can be separated and thus characterized using gel electrophoresis.
How do you determine the size of plasmid in gel electrophoresis?
The molecular weight size of unknown plasmids is determined by comparing their band pattern obtained in agarose gel electrophoresis with those obtained with plasmids that have been used as molecular weight or size standards.
How many bands should appear on the agarose gel when your plasmid is cut with both NHEI and Ecori?
Usually, a uncut plasmid should show at least three bands on gel. The top band most likely is the nicked/linearized plasmid.
Why are there 2 bands in the uncut lane?
However, it is likely that two or three bands will appear in the undigested plasmid lanes. The reason for this is that plasmids isolated from cells exist in several forms. One form of plasmid is called “supercoiled.” You can visualize this form by thinking of a circular piece of plastic tubing that is twisted.
Are plasmids linear or circular?
Plasmids are generally circular, but examples of linear plasmids are also known. These linear plasmids require specialized mechanisms to replicate their ends. Plasmids may be present in an individual cell in varying number, ranging from one to several hundreds.
Which of the following species have a linear plasmid?
NO+2 has a linear shape.