Download BIOT 3 Lecture 4 Gel Electrophoresis

Lecture 4
January 7, 2016
Biotech 3
Gel Electrophoresis
Factors Influencing DNA Migration
•
•
•
•
•
•
•
Size (length) – Reported as bp or Kb
Net charge
Shape (linear, circular, supercoiled, tertiary)
ssDNA or dsDNA
Bound protein or stain
Buffer system
Electrical field strength
Agarose Gels
• Large pore size
- larger samples >200 bp
• 0.3 – 0.6 % depending on DNA sizes (0.8% for our
plasmids)
• 3-4 mm thick
- thinner: better resolution
- thicker: more volume
Concentration of Agarose to Use
More fragile
Less fragile
What are the expected band sizes of the digested pET3a?
Which gel percentage would you choose?
Buffer Depth
• 3 – 5 mm over gel
- too little: may dry during run
- too much: band slowing, distortion, heating
• Buffer depletion (chamber design, run times)
Agarose Gel Buffers
(Continuous buffer system)
Continuous vs. Discontinuous Buffer Systems
Continuous buffer system
• composition of the buffer in the gels, wells and chambers are similar
• Gel pore size and molecular charge density are the only factors that have any effect on
stacking
• Limited in separating smaller molecules, smaller molecules have less of a difference
between their mobility
Discontinuous buffer system:
• Different buffer ions and pH in the gel and in the electrode reservoirs.
• Samples are loaded onto a non-restrictive large pore gel, called the “stacking” gel,
which overlays a smaller pore resolving gel
• Resolution is much greater
• Resolution is a direct result in the way samples concentrate into narrow zones during
migration through the gel)
• More on this topic when we discuss SDS-PAGE and protein separation
Sample Size
• Depends on fragments length, sizedistribution,
well volume, detection method, etc.
• Too small – not detected (10 ng DNA w/Ethidium
bromide, 10 ng protein w/ Coomassie)
Sample Loading Buffers
• Increases density (sucrose, glycerol)
• Adds color to the sample
• Adds mobility dye
- Bromophenol blue (front dye runs ~300 bp)
- Xylene cyanol (rear dye runs ~4 – 5 Kbp)
• May contain SDS, reducing agents, special loading
buffer
Examples
Overloaded
Comb removed
too soon
Short run
Bubble
Wrong TBE or TAE
concentration
Salt precipitate
in buffer
O'GeneRuler™ DNA Ladder 1kb