OhioMOd2013:Methods/gel electrophoresis

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Gel Electrophoresis

Gel Preparation

Prepare a 2% agarose gel (125 ml slab)

  • Weight 2.5 agarose into beaker
  • Fill up to 125 g with 0.5x TBE-Puffer
  • Boil in microwave until agarose is dissolved (2 min)
  • Some water may evaporate through heat. Add ddH2O until back up to 125 grams (apply water to sides of hot beaker)
  • Cool till hand warm
  • Add 1 ml of 1.375 MgCl2 solution
  • Add 7 μL ethidium bromid from 10 mg/ml stock solution. (use gloves for this protocol. minimize exposure)
  • Fill gel tray and install desired comb.
  • ONce solid, fill gel box with TBE/11 mM MgCL2 buffer.
  • Remove comb.

Gel loading

  • Sample: mix 12 μL of each DNA sample with 3 μL 6x loading dye. Load into lanes
  • Unfolded Plasmid: mix 1.2 μL of phage DNA (100 nM) with 10.8 μL ddH2O and 3 μL 6x-loading dye. Load into lanes
  • DNA ladder: add 6 μL of 1kb DNA ladder (New England Biolabs)

Run Gel

  • Put gel box into ice water bath
  • Apply constant 70 V across the gel.
  • Run for 3-4 hours.

Imaging

  • Can image using UV illuminator in dark room. Get picture>exposure>take picture>save under C: share files.

Crunch-n-Squeeze Purification

  • Use UV transilluminator for band visulaization, and cut out the desired band with razor bands.
  • Put slice of gel into tube.
  • Spin down the debris.
  • Cut off debris containing itp of the tub.

Invert tip into a freeze'n'squeeze spin column (Biorad).

  • Spin 12 min at 12,000G in tabletop centrifuge.
  • Throw away the top portion of the squeeze spin column.
  • Label and store purified DNA at 4°C
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