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