Horizontal Gel Electrophoresis for Nucleic Acids

1. Horizontal Gel Electrophoresis for Nucleic Acids Introduction for Restriction Enzyme Digest and Polymerase Chain Reaction Labs

2. How do we visualize DNA? • Agarose Gel Electrophoresis Complete a Gel Electrophoresis simulation at: http://gslc.genetics.utah.edu/units/biotech/gel/

3. Important Characteristics of DNA Structure • Nucleus is the “command center” • Chromosomes • Double-stranded • DNA helix • Encoded information in the form of bases = “genetic code”

4. Important Characteristics of DNA Structure • Genetic code: Guanine Adenine Cytosine Thymine • Phosphate-Sugar backbone NOTE • Phosphate groups are negatively charged • The mass:charge ratio is the same for all sizes of DNA fragments

5. 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

6. Components of an Electrophoresis System • Power supply and chamber, a source of negatively charged particles with a cathode and anode • Buffer,a fluid mixture of water and ions • Agarose gel, a porous material that DNA migrates through • Gel casting materials • DNA ladder, mixture of DNA fragments of known lengths • Loading dye, contains a dense material and allows visualization of DNA migration • DNA Stain, allows visualizations of DNA fragments after electrophoresis

7. Cathode - Anode + Buffer Power Supply Dyes Agarose gel

8. Power Supplies Bio-Rad’s Electrophoresis Equipment Precast Ready Agarose Gel

9. Electrophoresis Buffer • TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) are the most common buffers for duplex DNA • Establish pH and provide ions to support conductivity • Concentration affects DNA migration • Use of water will produce no migraton • High buffer conc. could melt the agarose gel • New Sodium Borate (SB) buffer allows gels to be run at higher voltages in less time than traditional buffers

10. 1% agarose 2% agarose Agarose Gel • A porous material derived from red seaweed • Acts as a sieve for separating DNA fragments; smaller fragments travel faster than large fragments • Plinko Model • Concentration affects DNA migration • Low conc. = larger pores better resolution of larger DNA fragments • High conc. = smaller pores better resolution of smaller DNA fragments

11. Loading Dye • DNA samples are loaded into a gel AFTER the tank has been filled with buffer, covering the gel • Contains a dense substance, such as glycerol, to allow the sample to "fall" into the sample wells • Contains one or two tracking dyes, which migrate in the gel and allow monitoring of how far the electrophoresis has proceeded.

12. DNA Staining • Allows DNA visualization after gel electrophoresis • Ethidium Bromide • Bio-Safe DNA stains • In gel staining

13. Agarose Gel DNA Fragments Frequent Misconceptions • Each band on the gel represents a single DNA strand...NOT TRUE. • A single band/position in a lane contains only one type of DNA sequence...NOT ALWAYS TRUE.