1. Gel Electrophoresis Lab

2. 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

3. 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

4. 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

5. 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

6. + - Power • DNA is negatively charged.
• 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

7. + - How fast will the DNA migrate? Power
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
DNA + -
Power
small
large
Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

8. Electrophoresis Equipment
Power supply
Gel tank
Cover
Electrical leads 
Casting tray
Gel combs

9. 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.

10. Running the Gel
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.

11. Cathode
(-)
DNA
(-) 
 wells
 CarolinaBromophenol Blue
Gel
Anode
(+)
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.

12. 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

13. DNA Marker Standard Curve
Size (bp) Distance (mm)
23, 9, 6, 4, 2, 2,

14. Applications Recombinant DNA Technology DNA Cloning
Constructing DNA Libraries
Southern Blot Hybridization