Gel Electrophoresis

What is Gel Electrophoresis? Electro- electricity -phoresis-to carry across A technique used to separate and analyze DNA and proteins Can be separated based on charge (positive vs. negative) or size The standard method for DNA is to use a gel that is made of agarose Agarose is a protein that is obtained from seaweed It is dissolved in boiling solution and then as it cools back down, it forms a gel

Why do we do it? After DNA is cut with restriction enzymes, there is a mix of DNA fragments of all different sizes Review: What is a restriction enzyme? Using gel electrophoresis, the DNA can then be separated by size and further analyzed This is used for: Sequencing and analyzing a DNA strand DNA fingerprinting Diagnosis of genetic disorders

How does it work? DNA is a negatively charged molecule The gel is exposed to an electrical current and the negatively charged DNA molecules will move towards the positive electrical charge The gel is like a sponge in the sense that there are pores (holes) in the gel for the DNA to travel through

The speed that the DNA moves is dependent on the size of the piece of DNA The smaller DNA fragments move faster: they have an easier time navigating through the holes and moving down the gel and will finish closer to the positive end The larger DNA fragments move slower, ends closer to the negative

How is it set up? Once the agarose has been dissolved, it is poured into a mold to settle While still in the liquid form, a comb is placed into the gel Once the gel is hardened and the comb is removed, there are wells, or holes, that the DNA can be put into The gel is placed into a solution (buffer) that will help conduct the electricity

Gel Mold and Combs

Electrophoresis Setup DNA buffer   wells Positive  End  Negative End

Preparing the DNA DNA is mixed with dye (so it can be seen while loading) and glycerol (to make it sink)

Loading the DNA into the Gel DNA is added to the wells Carefully added to the well with a pipette so that a hole is not poked in the gel The DNA should sink to the bottom

Electricity is applied to gel and the DNA begins to move

~~~~~~~~~~~~~~~~~~~~~~~~ Agarose gel Negative Positive DNA fragments buffer ~~~~~~~~~~~~~~~~~~~~~~~~ Negative Positive current buffer ~~~~~~~~~~~~~~~~~~~~~~~~

Gel Electrophoresis Video

How is the DNA measured?  100  200  300  1,650  1,000  500  850  650  400  12,000 bp  5,000  2,000 DNA migration Usually at least one well of the gel will be loaded with a known DNA sequence called a marker Acts as a ruler for DNA fragments Unknown DNA fragments can then be compared to this marker band, or DNA ladder, to figure out the size

How is the DNA seen? After the gel is finished running, it must be stained so that DNA can be seen by the naked eye Individual DNA strands cannot be seen, but groups of DNA fragments of the same size can Brighter/darker bands indicate more DNA * The original dye separates from the DNA strands during the electrophoresis process