1. Lecture 4
January 7, 2016
Biotech 3

2. Gel Electrophoresis

3. 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

4. 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

5. 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?

6. 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)

7. Agarose Gel Buffers (Continuous buffer system)

8. Continuous vs. Discontinuous Buffer Systems
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

9. 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)

10. 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

11. Wrong TBE or TAE concentration Salt precipitate in buffer
Examples
Overloaded
Comb removed too soon
Short run
Bubble
Wrong TBE or TAE concentration
Salt precipitate in buffer

12. O'GeneRuler™ DNA Ladder 1kb