Bitan:Agarose gel electrophoresis

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Four μl PCR reaction-mix are analyzed using a 0

Agarose electrophoresis of DNA product

  1. Analyse 4 μl PCR reaction-mix using 0.8% agarose gel electrophoresis and 5× RapidRun Loading Dye (1 μl, 77524, usb) to verify whether sufficient amount of DNA was produced. 
  2. Agarose gel is prepared in 1× TBE (Tris-borate-EDTA) buffer:
    1. Prepare 10× TBE:
    2. Tris base                                 108 g
    3. Boric acid                                55 g
    4. EDTA (0.5M, pH 8.0)               40 ml
    5. Make up to 1 L with autoclaved deionized water.
  3. Prepare agarose gel:
    1. Agarose (<1000 BP)                1.6 g
    2. 1× TBE                                     200 ml
    3. Microwave at 1-min intervals until completely dissolved.
    4. Release the pressure inside the agarose bottle after each 60-sec interval in a fume hood.
    5. After cool down (~50 °C), add ethidium bromide (EtBr); the stock is 10 μg/μl (1% solution)                            10 μl (0.05% final concentration)
  4. Assemble the gel mould. In the fume hood, pour agarose up to about half the height of the teeth of the gel comb.  Take extreme care when handling ethidium bromide (EtBr) and EtBr-containing gels.  Work in the hood.
  5. Immerse gel in running buffer (1× TBE buffer) in the running chamber, place colored tape under lanes to help visualization of the wells (if desired)
  6. Load the gel wells with samples taking note of the loading order.  Make sure the gel end with lanes is towards the black electrode (cathode). Run at 100V for 15–20 min.  Visualize DNA under UV and take a picture using Spencer Lab’s camera.

 

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