文档介绍：Gel Electrophoresis of DNA
What is Gel Electrophoresis?
Electro = flow of electricity, phoresis, from the Greek = to carry across
A gel is a colloid, a suspension of tiny particles in a medium, occurring in a solid form, like gelatin
Gel electrophoresis refers to the separation of charged particles located in a gel when an electric current is applied
Charged particles can include DNA, amino acids, peptides, etc
Why do gel electrophoresis?
When DNA is cut by restriction enzymes, the result is a mix of pieces of DNA of different lengths
It is useful to be able to separate the pieces - . for recovering particular pieces of DNA, for forensic work or for sequencing
What is needed?
Agarose - a haride made from seaweed. Agarose is dissolved in buffer and heated, then cools to a gelatinous solid with work of crosslinked molecules
Some gels are made with acrylamide if sharper bands are required
Buffer - in this case TBE
The buffer provides ions in solution to ensure electrical conductivity.
Not only is the agarose dissolved in buffer, but the gel slab is submerged (submarine gel) in buffer after hardening
Also needed are a power supply and a gel chamber
Gel e in a variety of models, mercial through home-made, and a variety of sizes
How does it work?
DNA is anic acid, and is negatively charged (remember, DNA for Negative)
When the DNA is exposed to an electrical field, the particles migrate toward the positive electrode
Smaller pieces of DNA can travel further in a given time than larger pieces
A gel being run
Agarose block
Positive electrode
DNA loaded in
wells in the agarose
Black background
To make loading wells easier
Comb
Buffer
Steps in running a gel
DNA is prepared by digestion with restriction enzymes
Agarose is made to an appropriate thickness (the higher the % agarose, the slower the big fragments run) and ‘melted’ in the microwave
The gel chamber is set up, the ‘comb’ is inserted
The agarose may have a DNA ‘dye’ added (or it may be stained la