Wittrup: Gel electrophoresis

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Materials

  • Restriction stop buffer (10x) or sample loading buffer (6x)
  • 10x TAE (Invitrogen 15528-026)
  • Agarose
  • DNA samples
  • DNA ladder: 1 kb (Invitrogen 15615-016) or 100 bp (Invitrogen 15628-019)
  • SYBR Gold DNA Stain (Invitrogen S-11494)
  • DNA gel box

Restriction Stop Buffer (10x)

  • 7.5 mL glycerol
  • 3 mL EDTA (0.5 M, pH 8.0)
  • 0.15 g sodium dodecylsulfate
  • 0. 015 g bromophenol blue
  • to 15 mL ddH2O

Sample Loading Buffer (6x)

  • 3 mL glycerol
  • 0.025 g bromophenol blue
  • 0.025 g xylene cyanol
  • to 10 mL ddH2O

Protocol

Prepare Gel

1. Prepare gel box: place tray with two side handles in rectangular gel box; add comb(s) with appropriate number of wells.

2. Add 50 mL of 1x TAE to a flask (this is for a small gel; larger gels require more).

3. Add appropriate amount of agarose (e.g., 0.50 g for a 1% gel).

4. Microwave flask until liquid boils (~40 s; watch carefully to avoid a spill). Swirl flask gently to mix.

5. Pour slowly into case. Allow gel to soldify (~30 min).

Prepare Samples

1. Combine ddH2O, buffer (either restriction stop buffer or loading buffer), and DNA.

Examples:

DNA Ladder: 0.5 μL of DNA ladder (1 μg/μL), ddH2O, 4.5 μL ddH2O, 1 μL 6x loading buffer

DNA Digestion: 18 μL of restriction digested DNA, 2 μL 10x restriction stop buffer.

Complete and Run Gel

1. When the gel has solidified, remove it from the gel box and place on upper level of electrophoresis apparatus. Remove comb.

2. Add 1x TAE to cover gel.

3. Pipette samples into the wells.

4. Cover case with red (-) electrode away from samples.

5. Apply constant voltage (70-100 V).

6. Allow the gel to run desired length. (bromophenol blue dye moves with 500 bp DNA)

Stain

Use caution: SYBR Gold binds DNA

1. Thaw SYBR Gold at room temperature (from 10 μL aliquot at -20º).

2. Add 40 mL of 1x TAE to container.

3. Add 10 μL of SYBR Gold to container. Mix well.

4. Place gel in container.

5. Incubate with rocking for >10 min. Shield from light for long incubations. (Stain may be reused if desired)

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