Gel Electrophoresis Lab

Agarose Gel Electrophoresis

• Electrolysis: the splitting of water using electricity– current splits water into hydrogen ions (H+) and

hydroxyl ions (OH-)

• Electrophoresis: a method of separating charged molecules in an electrical field; DNA has an overall negative charge

• Used to separate DNA fragments by size

Agarose Gel Electrophoresis

•We will be using agarose gel electrophoresis to determine the size of DNA fragments made by using 2 different restriction enzymes on lambda DNA-Hind III and EcoRI. We will run lambda DNA without restriction enzymes as well.•3 vials

• Lambda DNA• Lambda DNA cut with EcoRI• Lambda DNA cut with HindIII

Lambda Phage DNA

Genomic DNA of a bacterial virus Attacks bacteria by inserting its nucleic acid into the host

bacterial cell Replicates rapidly inside host cells until the cells burst

and release more phages Harmless to man and other eukaryotic organisms

What is needed for restriction digestion?

• Template DNA, uncut DNA, often bacterial phage DNA (Lambda DNA)

• Restriction enzyme(s), to cut template DNA • Restriction Buffer, to provide optimal

conditions for digestion• Water Bath

• DNA is negatively charged.

+-

Power

DNA

• When placed in an electrical field, DNA will migrate toward the positive pole (anode).

H

O2

• An agarose gel is used to slow the movement of DNA and separate by size.

+-

Power

DNA

How fast will the DNA migrate?strength of the electrical field, buffer, density of agarose gel…• DNA is negatively charge due to the phosphate groups. This will cause the DNA to migrate to the positive electrode• Migration is based on the size of the DNA• Small DNA move faster through the agarose gel than large DNA…gel electrophoresis separates DNA according to size

smalllarge

Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

Casting tray

Gel combs

Power supply

Gel tank

Cover

Electrical leads

Electrophoresis Equipment

Loading the Gel

Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.

Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.

Running the Gel

wells CarolinaBromophenol Blue

Cathode(-)

Anode(+)

Gel

After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.

DNA(-)

Actual Results of Restriction Enzyme Digestion

• Lane 1, DNA markers (HindIII lambda digest)

• lane 2, uncut lambda DNA

• lane 3, lambda DNA digested with PstI

• lane 4, lambda DNA digested with EcoRI

• lane 5, lambda DNA digested with HindIII

DNA Marker Standard Curve

• Size (bp) Distance (mm)• 23,000 11.0• 9,400 13.0• 6,500 15.0• 4,400 18.0• 2,300 23.0• 2,000 24.0

Applications

• Recombinant DNA Technology• DNA Cloning• Constructing DNA Libraries• Southern Blot Hybridization