PreparationofAgaroseGelsforDNAseparations

Weigh out the desired amount of agarose and place in an Erlenmeyer flask with a measured amount of electrophoresis buffer, e.g. for an 0.8% gel, add 0.8 gm of agarose and 100ml of TBE Buffer (1X), to a 200 ml flask. The larger flask insures against the agarose boiling over. Dissolve the agarose in a boiling water bath or in a revolving-plate microwave oven. All the grains of agarose should be dissolved and the solution clear. Cool the solution to 60°C (70°C for concentrations 2% or above) and pour immediately. Allow the gel to set for one-half hour before using. Make sure to use the same electrophoresis buffer in the gel as for the running buffer.

DNA Size Separation by Agarose Concentration 
 

Prepare sample by adding concentrated DNA loading buffer. DNA always migrates towards the positive electrode. Bromophenol blue (BP) generally co-migrates with 0.5 kb fragments of DNA. Stain with 0.5 mg/ml ethidium bromide for 10 minutes.

• Acrylamide is used at a 19:1 ratio with bis-acrylamide, filtered and refrigerated.

• For 40 ml of gel use 25 ml of TEMED and 250 ml of 10% APS solution to polymerize.

• Prepare sample by adding concentrated DNA loading buffer.

• DNA always migrates towards the positive electrode.

• Stain with 0.5 mg/ml ethidium bromide for 10 minutes.

  
  DNA Size Separation by Acrylamide Percentage

  DNA Size Range (Base Pairs)Acrylamide (%)100 - 1,0003.580 - 5005.060 - 4008.040 - 20012.010 - 10020.0

  DNA Mobility in Acrylamide Gels

  Migration of marker dyes in bp for native polyacrylamide non-denaturing gels 
   

  Gel %	Bromophenol blue (BP)	Xylene cyanole (XC)
  3.5	100	460
  5.0	65	260
  8.0	45	160
  12.0	20	70
  20.0	12	45

  Migration of marker dyes in bp for polyacrylamide denaturing gels 
   

  Gel %	Bromophenol blue (BP)	Xylene cyanole (XC)
  5.0	35	130
  6.0	26	106
  8.0	19	75
  10.0	12	55
  20.0	8	28