Gel Electrophoresis

Overview

The following protocol covers the basics of pouring an agarose gel and running a DNA product.

Protocol

• Choose the concentration of agarose appropriate for the DNA you are trying to separate. In general, a 1% gel will work fine.

Gel concentration based on DNA size

• Mix Agarose into TAE to get the appropriate concentration and volume depending upon tray size. Use Low EEO grade agarose. It is possible to just use TE also.
• Microwave the mixture. Generally 1 minute for every 100 ml.
• Remove from microwave and let cool slightly
• Add Ethidium Bromide to gel at a concentration of 0.5 µg/ml
• Allow gel to cool more. If it is too hot, the seals on the gel tray will leak.
• Pour Agarose into gel tray and place combs at appropriate distances.
• Let cool until hardened.
• Place gel tray in running box and cover gel with TAE.
• Load wells with DNA mixed with loading dye. Load one well with a DNA ladder. Generally small wells hold up to 20 µl, while larger wells hold up to 50 µl
• Cover electrophoresis chamber and plug into a power box. Turn on power generally at 100 to 300 volts (supposed to have 1 to 5 V/cm of gel, measuring length of gel from anode to cathode). IF the power is too high, the gel will melt.
• Watch gel and turn off power based on how far the loading dye has travelled.
• Note: If you forget to put Ethidium bromide in the gel, the gel can be bathed in TAE containing Ethidium bromide for 20 minutes and then imaged.