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
for Linear Fragments (kb) | (%) |
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 Gel for Small DNA Fragment Separations
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
Migration of marker dyes in bp for polyacrylamide denaturing gels
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