Speed Buffer for DNA Gel Electrophoresis

Speed Buffer for DNA gel electrophoresis

Use Speed Buffer instead of Tris-acetate-EDTA (TAE) or Tris-borate EDTA (TBE) buffer. This buffer allows you to run a DNA gel at high voltages without overheating and melting your gel. Instead of 100 volts for 45-60 minutes, you can electrophorese at 350 volts for 10-15 minutes.

A 20X concentrate stock is diluted to 1X and then used to make and run the gel, just as was done using TAE. Run the gel using “constant voltage” and 350 volts.

20X Speed Buffer

0.2 M NaOH

stir in boric acid until pH is 8.0

Store at room temp.

Pouring an agarose gel for DNA gel electrophoresis

Make 1 liter of 1x buffer:

• Measure 50 ml of 20X Speed buffer into 1 liter graduated cylinder
• Add water from distilled water tap to 1 liter
• Transfer the buffer to our 1x Speed Buffer container.
• Wearing gloves, add 25 µl of *ethidium bromide* (EtBr, 10mg/ml stock) – located in refrigerator.
• Replace cap and swirl buffer to mix.

Prepare 1% (w/v) agarose in 1X Speed Buffer: (Agarose mass and buffer volumes can be adjusted as needed for different concentrations and number of gels.)

• Weigh out 0.5 g of agarose powder
• Add powder to 50 ml of 1X buffer in a 250 ml flask. (Use a larger flask for larger gel volumes.)
• Microwave 3 minutes, until boiling.
• Using the silicon ‘hot glove’ CAREFULLY remove the flask. Gently swirl to mix, checking that the agarose is completely dissolved and no ‘cubes’ are floating in the solution.
• Allow the agarose to cool for 5-10 minutes.
• Assemble gel box during this time, placing the gel tray crosswise in gel box so the rubber gaskets make a seal.

Pour and run the gel:

• Choose the appropriate comb for the number of samples and ladders you need to load.
• Pour in the agarose into the tray, then place the comb(s) in the desired spaces.
• Allow gel to solidify for about 10 minutes.
• Gently remove combs; turn gel tray so the DNA “runs to the red” electrode.
• Fill up gel box with 1X Speed Buffer with EtBr buffer to just above the gel.
• Add Loading dye to your samples, if necessary, and load each sample in a well.
• Include a DNA ladder for each row of wells.
• Put on the gel cover and run the gel at 300 volts for 12-15 minutes.
• Visualize the gel on UV gel box or using the GelDoc in Cell lab.
• Discard gel in biohazard bag.

**Ethidium bromide is a potential carcinogen and should be handled with care. Wear gloves when handling solutions and contaminated equipment. Remove gloves when touching non-electrophoresis related items and equipment, especially personal items and computers.