Agarose Gel Electrophoresis-Protocol

Agarose Gel Electrophoresis

Caution: wear gloves because of Etedium Bromide(Cancer Causing).

1. Dilute the 10X running buffer (TBE with EtBr) to 1X. Calculate the amount of the 1X buffer in gel tray(s). Add appropriate amount of agarous (for 5 mm standard gel; 0.7 % or 1 %) in the buffer, melt in the microwave oven. Pour the melting agarous (appr. 40-50 C) into a graduate cylinder, add 1X buffer up to the pre-determined volume, pour back into the flask to mix, and fill the gel tray (watch out bubble). Let set at least one hour to harden.

    Gel size (W X L) Agarous (Rec)  
    Gel tray   (cm X cm)  Owl  Rec. Buffer (L)  1.0 %   0.7 %
    
    A3    20 X 40   600  400  3.0   L 4.0   2.8 
   
    B2    12 X 14   130  100  0.5   L 1.0   0.7
   
    C1   7.6 X 5.1   30   20  0.1   L 0.3   2.7
    
   

   
   
2. Fill an electrophoresis chamber with 1X running buffer until the gel is covered by a couple of millimeters.
   
3. Adjust DNA concentration of sample (appr. 5 to 8 �g genomic DNA; 0.5 �g plasmid DNA per lane).
   
4. Load the DNA samples (with 1/10 volume of tracking dye and heat shocked for 5 min at 65) carefully with a Pipetman P20 by slowly expelling the solution into a well, with the pipet tip slightly below the top of the well. Do not hold the pipet too far in the well: the sample will sometimes come out the bottom. Try not to plug the pipet tip against the side of the well either: the sample will usually squirt out when there gets to be enough pressure from the pipetman.
   
5. Load the appropriate marker DNA flanking the sample lanes.
   
6. Close the lid on the electrophoresis kit. Always run the DNA toward the red (+) terminal. Electrophoresis the gel at 11 volts overnight or 100 volts for a couple of hours.
   

   Note: the size of DNA to be separated needs to be matched to the agarous concentration:

   Agarose %	Range of separation
      0.3		60 - 5 kb
      0.6		20 - 1 kb
      0.7		10 - 0.8 kb
      0.9		 7 - 0.5 kb
      1.2		 6 - 0.4 kb
      1.5		 4 - 0.2 kb
      2.0		 3 - 0.1 kb