Gel Electrophoresis: Main Principles • Separate DNA fragments by size • Smaller DNA fragments move faster • Run a “size marker” to compare size of separated fragments • DNA moves through gel due to electric field • DNA is negatively charged • Moves towards positive charges • Visualize separation • Use nucleic acid stains such as Gel Red or ethidium bromide • When excited under UV light transmits at visible wavelength
Smaller Fragments Move Farther 100bp size marker
Step 1: Pouring a gel • Need to create gel of appropriate agarose concentration • Combine agarose (powder) • TBE (liquid buffer) • Microwave to dissolve agarose • Add stain and swirl to mix • Gel Red • Pour into gel bed • With a comb!
Preparing a 2% agarose gel with 50mL 1x TBE • Weigh out 1g agarose in weight boat • Add 50ml of 1x TBE • Needs to be diluted! • Cover with saran wrap • Poke holes! • Microwave til it boils • Careful it’s hot! • Allow to cool to room temperature • Add 5ul Gel Red • Swirl to mix • Pour into gel bed • Insert comb • Let solidify
Loading a gel • This is easy but takes practice • So practice! • The gel will be submerged in buffer • Tip does not need to go INTO well • Hold tip over well and release! • Loading dye is heavy it will fall into place • Try not to poke holes • Make a list of what sample goes where BEFORE loading gel • Make sure we are loading at the NEGATIVE end
Running a gel DNA needs electric field to move Power source supplies this electric field Set power source to the voltage you want and let run for specified amount of time Make sure positive goes to positive and negative to negative Make sure wells are at negative end!
Loading and Running a Gel • Take out comb • Make sure wells are at negative end • Fill gel rig with 1x TBE buffer • Enough so wells are well covered • Make a list of what sample will go where • Load: • 20 µL of pBR322/BstNI size markers • 10 µL of the undigested (U) sample/loading dye mixture • 16 µL of the digested (D) sample/loading dye mixture • Carefully release this while holding tip OVER the well • Sample will fall into well • When finished put lid on gel bed (+/+ and -/-) • Hook up to power source • Set power source to 200v and start • Check in- did the bands move?
Visualizing Gels • Gel Red attaches to DNA • Intercalates between base pairs • “Slips in” • This strips away water • Gel Red glows under UV light • Removal of water during intercalation allows it to glow brighter
Analyzing Results • Looking at banding patterns • Bands present? • Number of bands per each lane? • Relative size of bands • Using 100bp ladder
Visualizing Gels • Turn off power source • Pour running buffer back into container • Take gel out of gel rig • Be careful not to break it! • Bring to UV transluminator • Visualize • Make sure you are wearing a shield! • Take photos and analyze