Lecture 4 January 7 2016 Biotech 3 Gel

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) ss. DNA or ds. DNA 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 p. ET 3 a? 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 p. H 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'Gene. Ruler™ DNA Ladder 1 kb