GEL ELECTROPHORESIS

What is Gel Electrophoresis?

Electro- electricity

-phoresis-to carry across

A technique used to separate and analyze DNA and proteinsCan be separated based on charge (positive vs. negative) or

size

The standard method for DNA is to use a gel that is made of agaroseAgarose is a protein that is obtained from seaweedIt 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 sizesReview: 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 strandDNA fingerprintingDiagnosis 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 DNAThe 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 settleWhile still in the liquid form, a comb is

placed into the gelOnce 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

buffer

Negative End

Positive End

DNA

wells

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 PositiveDNA fragments

buffer~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~

Negative Positive

current

buffer~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~

~~~~~~~~~~~~~~~~~~~~~~~~

Gel Electrophoresis Video

How is the DNA measured?

Usually at least one well of the gel will be loaded with a known DNA sequence called a markerActs 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

100 200 300

1,650

1,000

500

850

650

400

12,000 bp

5,000

2,000DNA

migration

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 canBrighter/darker bands

indicate more DNA