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University of Washington School of Medicine, Department of Microbiology     

Gel Electrophoresis

Note: There are a myriad number of gels and techniques used in electrophoresis. For exact gel recipes and techniques, consult appropriate manufacturer's manuals.

Note: Gel Electrophoresis is run at high voltages. Do not run an electrophoretic experiment around water. Do not touch the gel once the power has been running. Touching the gel when running runs the risk of electrocution.

Note: There is a danger of chemical burns when mixing the various gel components together. Always wear gloves (latex), glasses, and lab coats when dealing with gels and electrophoresis.

Assembly

1. Assemble the glass sandwich first. The two pieces of glass have to sandwich two spacers of identical thickness. Both sides of the glass have to be flush with one another, otherwise the glasses can not be clamped together. Use guides and clamps (see individual manufacturer's parts) to hold the sandwich together. Place sandwich in holder.

2. A popular gel stack is called Laemmli (Nature227,6801970) where two gels are put together. The first gel (least downrange) is a stacking gel where the components are concentrated together for between band resolution. The second gel (most downrange) is a separating gel, which splits the different bands apart for visualization and recovery. See manual for explicit details on how to properly prepare and cast gels. Common gels are SDS, agarose, and Laemmli. In order to remove air bubbles from the gel, use a comb (tilted 10 degrees relative to the parallel gel). 

3. There are three main methods for loading samples in the wells.
a. Loading liquid samples into wells formed by a well-cast comb is a popular method. Prepare 1500ml of electrode buffer. Place 1150 ml onto the lower buffer chamber Place the cooler core (usually filled with antifreeze or 20/80 methanol and water) into the lower buffer chamber at a slight angle to prevent air bubbles. For 16cm plates, dilute the lower buffer with distilled water to a level 1cm above the bottom of the plate. Mix the lower buffer well. Pour 325-350ml of electrode buffer (undiluted) into the upper chamber. Load the samples into the wells under the electrode buffer with a Hamilton syringe with 1-2mm between the end of the syringe and the well bottom. 

b. You can load the preparative run of samples on a flat bed of gel. See manual for details. 

c. You can use gel itself as a sample, as in 20D procedures. See manuals for details.

4. Place the lid on the electrophoretic box correctly. There is only one way this can be done in order not to reverse the anode and cathode ends. 

5. Attach the lead to a power supply (see Power Supplies). When everything is set, turn on the power (Output Supply to ON). DO NOT TOUCH GEL WHEN POWER IS ON. Running conditions (i.e. amps and voltage) are dependent on a varied number of conditions including gel composition and thickness. 

6. After electrophoresis is compete, turn off power supply and disconnect the leads and coolant hoses. Pour off the upper buffer. 

7. Remove the gel by either grasping the two corners and pulling away or by placing both the gel and plate in an agitating fixative solution until the gel separates. Use the fixative (usually a mixture of 40% methanol and 10% acetic acid).

Maintenance

1. Rinse the core, the chamber, and the clamps (i.e. nothing touching the gel itself) with distilled water after every use.

2. Wash the glass plates, spacers, and combs with a laboratory detergent and then rise fully with distilled water. If this does not clean the plates, soak the plates in a strong acid solution (i.e. chromic acid/sufuric acid) for thirty or more minutes and rinse fully with distilled water. 

3. If glass tubes are used in electrophoresis , rinse with lab detergent, scrub out, and rinse with distilled water. Store the glass tubes in chromic/sulfuric acid solution until next use. Rinse with water and force air dry before next use. 

 

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jl, 9may04

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