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