This article describes the process of analyzing DNA amplicons resulting from multiplex polymerase chain reaction (PCR) using agarose gel electrophoresis. The methodology includes preparing the gel, loading samples, and visualizing the results under UV light.
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.
According to the manufacturer's recommendations, add agarose gel powder in 1x TBE buffer to reach 1.5 (weight/volume) percent, and heat to dissolve.
Prior to casting, tilt the dissolved gel solution briefly under running water. Add 5 microliters of fluorescent nucleic acid dye per 50 microliters of gel solution, and mix.
Assemble and level the casting tray. Add combs as appropriate for the number of samples. Pour the gel solution into the cast, and wait for it to solidify.
After that, submerge the cast containing the gel in 1x TBE buffer in a gel electrophoresis system. Mix 5 microliters of the PCR products together with 2 microliters of loading dye in new PCR strips. Transfer 5 microliters of the mixtures to the wells in the gel. Save at least one well empty. Add 5 microliters of DNA ladder in the empty well for reference.
Plug in the electrodes and run the gel at 110 volts per 15 centimeters for approximately 1 hour. Use a UV-based gel imaging system to verify the presence of multiple bands representing PCR amplicons.
繁體中文
