gel electrophoresis lab. agarose gel electrophoresis electrolysis: the splitting of water using...
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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