Image by D. The agarose obstacle course The DNA race occurring in an electrophoresis gel is fair but it is an obstacle course. Previous Page Next Page. Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like DNA , RNA and proteins according to their size. Charged molecules move through a gel when an electric current is passed across it. An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge.
The movement of charged molecules is called migration. Molecules migrate towards the opposite charge. A molecule with a negative charge will therefore be pulled towards the positive end opposites attract!
The gel consists of a permeable matrix, a bit like a sieve, through which molecules can travel when an electric current is passed across it. Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance.
As a result the molecules are separated by size. DNA is negatively charged, therefore, when an electric current is applied to the gel, DNA will migrate towards the positively charged electrode. Shorter strands of DNA move more quickly through the gel than longer strands resulting in the fragments being arranged in order of size.
The use of dyes, fluorescent tags or radioactive labels enables the DNA on the gel to be seen after they have been separated. They will appear as bands on the gel. A DNA marker with fragments of known lengths is usually run through the gel at the same time as the samples. Ethidium bromide is a sensitive, easy stain for DNA. So loading buffer provides one more function in gel electrophoresis. Loading buffer also increases the density of the sample.
Recall that denser objects sink, so adding loading buffer to the DNA samples will enable the DNA molecules to sink into the wells in the gel in preparation for gel electrophoresis. The phosphate molecules that make up the backbone of DNA molecules have a high negative charge. When DNA is placed on a field with an electric current, these negatively charged DNA molecules migrate toward the positive end of the field, which in this case is an agarose gel immersed in a buffer bath. Gel electrophoresis is one of the techniques scientists use to look at the DNA they have.
This technique separates DNA molecules by size. First a gel is prepared. Gels are made of agarose , a seaweed extract similar to gelatin. Polymerase chain reaction PCR is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving. Restriction enzymes are enzymes isolated from bacteria that recognize specific sequences in DNA and then cut the DNA to produce fragments, called restriction fragments.
Restriction enzymes play a very important role in the construction of recombinant DNA molecules, as is done in gene cloning experiments. Gel electrophoresis. DNA fragments are separated according to their size. Proteins can be separated according to their size and their charge different proteins have different charges. The negatively charged DNA can be pulled toward the positive field of the gel.
The smallest fragment of basepairs 1 is hardly visible, while the biggest fragment of more than Band 3 contains smaller DNA fragments than band 2, but is still much brighter. This is because there is more nanograms of DNA in 3 than in 2 the number of molecules in 3 must be much higher than in 2. 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.
In vivo, plasmid DNA is a tightly supercoiled circle to enable it to fit inside the cell. In the laboratory, following a careful plasmid prep, most of the DNA will remain supercoiled, but a certain amount will sustain single-strand nicks. Given the presence of a break in only one of the strands, the DNA will remain circular, but the break will permit rotation around the phosphodiester backbone and the supercoils will be released.
A small, compact supercoiled knot of ccc-DNA sustains less friction against the agarose matrix than does a large, floppy open circle of oc-DNA. Linear DNA runs through a gel end first and thus sustains less friction than open-circular DNA, but more than supercoiled.
Thus, an uncut plasmid produces two bands on a gel, representing the oc and ccc conformations. If the plasmid is cut once with a restriction enzyme, however, the supercoiled and open-circular conformations are all reduced to a linear conformation. Following isolation, spontaneous nicks accumulate as a plasmid prep ages.
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