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 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

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

O'GeneRuler™ DNA Ladder 1kb