Agarose Gel Electrophoresis of DNA Amplicons Post PCR: A Method to Analyze Products of Multiplex PCR and Evaluate PCR Reaction Success
Transkript
To analyze DNA amplicons – amplified DNA fragments of specific length – resulting from multiplex polymerase chain reaction, PCR, using agarose gel electrophoresis, assemble a casting tray with a comb to create wells for sample loading.
Pour an appropriate concentration of molten agarose, a linear polysaccharide, dissolved in electrophoresis buffer, supplemented with a fluorescent DNA dye, onto the casting tray and allow to solidify.
The molten agarose polymerizes, forming a three-dimensional gel with microscopic pores.
Place the casted gel in an electrophoresis tank, with the comb-side oriented near the negative cathode. The tank contains the electrophoresis buffer used in gel preparation to maintain optimal conductivity. Remove the comb.
Transfer the PCR product sample, mixed with loading dye solution to monitor sample migration, into the wells. Load one well with DNA ladders of defined length. Initiate electrophoresis.
DNA amplicons, with evenly spaced negative phosphate groups, migrate from the wells into the gel towards the positive anode and get separated based on their length due to high mobility of shorter amplicons than larger ones. Simultaneously, the positive fluorescent DNA dye in the gel migrates in the reverse direction and combines with DNA amplicons, intercalating between the bases.
Post electrophoresis, image the gel using ultraviolet light. For a successful PCR, the dye-intercalated DNA amplicons appear as discrete, fluorescent bands closest to the ladder of expected amplicon length. The amplicon yield can be estimated through fluorescence intensity.
