Agarose Gel Electrophoresis

To check PCR products, restriction digests, etc..  Generally use a 1% gel.  For separating fragments that are 500bp or smaller, use 2% agarose. If the downstream application is DNA extraction, use 0.7% agarose.

  1. Pour a 1% gel.  Volumes are:
    • Smalllest gel box (blue) = 20ml; 0.2g agarose
    • Medium gel box (orange) = 50 ml; 0.5g agarose
    • Large gel box (green/clear) = 100ml; 1g agarose

Melt the agarose in 0.5X TBE in the microwave at 40% power until all agarose pellots are dissolved.  Cover flask. 

Add GelRed at a dilution of 1:10,000 in melted agarose. Ex. Add 5 ul of GelRed to 50 ml melted agarose solution. 
This step can be done while gel is still hot

Pour melted agarose solution into gel box mold (turn gel holder sideways in gel box to create mold). Insert plastic comb for proper number of lanes.  Allow the gel to dry for about 15 to 20 minutes.  

Remove comb, turn gel holder, and add 0.5x TBE buffer, making sure that the gel is completely submerged (do not fill past max fill line).

B. Load the gel.
  • Load 10ul of ladder into Lane 1 (already contains loading buffer)
    • DNA Ladders: We have 2 ladders.  Sty I ladder is for larger fragments; NEB 100 bp ladder is for smaller fragments.  See StyI ladder picture next to gel boxes for a reference
  • Each sample needs loading buffer, which should be used at a ratio of 1:6.  Loading buffer is used as a marker for migration rates and also to give the sample density so that it sinks to the bottom of the well. Loading buffer can be added directly to sample, or 1 ul loading dye can be pipetted onto parafilm for each sample you have,  then mixed with 5ul DNA prior to loading.

Small-tooth comb wells can hold about 15-20ul

    • Larger-tooth comb wells can hold about 50ul.  This varies with how thick the gel is.  
    • If larger wells are needed, you can tape two teeth.  This is sometimes useful for gel extractions.

After all samples and ladder(s) have been loaded, slide/place the lid on the gel box, make sure it is plugged into the powersource, and turn the powersource on.  Run it at about 92 volts for 60-90 minutes, or until loading buffer bands are where you want them.  If you want to run it slower, turn the voltage down.  Your fragments will run off if you run it too long.  Use the loading buffer bands as a marker!

  1.  Check the gel when it is done using the gel box in Manter common room. If it's not spread out enough, run longer.  Remember to turn off the power when you disconnect the lid from the box!  Also, when not in use, please make sure the lid is on to minimize evaporation of buffer and to keep dust out.  Gel can be thrown in the trash when done