Agarose Gel Electrophoresis

From 2012.igem.org

Gel Electrophoresis

For the analysis of PCR-amplified products, gel electrophoresis is the method of choice. Thereby, the DNA is separated by charge and size. The PCR samples are run on agarose gels with different percentages according to the product sizes applying current. Later on, these fragments within the gel are examined under UV light to ensure the DNA fragment length synthesized in the PCR reaction. Prior to UV analysis, a staining method of the DNA, here using ethidium bromide (EtBr), is mandatory.

Pouring the Gel

1% agarose gels are standard to separate DNA. The percentages and thus the degree of polymerisation of the gel influences the degree of separation, i.e. PCR products of similar size can be distinguished by applying a percentage lower than <1%. To pour a 1% agarose gel weight 1 g of Ultra Pure Agarose for every 100 ml of 1x TAE buffer. For preparation of 1x TAE buffer fill 10 mL of 50x TAE buffer stock ad 500 mL ddH2O. Ensure that the 1% agarose in 1x TAE buffer is boiled throughly and dissolved completely without streaks. Incomplete boiling will bias the separation results. The liquid is poured in a gel tray with the wanted comb which are assembled in a holder. After solification the gel is placed in a gel chamber and is fully covered with 1x TAE buffer post removal of the comb.

Loading the Gel

Prior to loading, the DNA samples are homogenized with 6x Loading Dye (LD) resulting in a 1x final concentration of the LD (4 μl 6x LD ad 20 μl PCR reaction). Be aware of the fact, that PCR samples should not be mixed with LD if further experiments with the samples are needed to be done. If this is the case, mix the PCR samples with LD on parafilm. Meanwhile, the PCR samples are kept on ice. The 1 kb DNA ladder is stored in the 4°C fridge and is suitable for larger PCR products ranging from 250 bp to 10000 bp (GeneRuler 1 kb DNA Ladder (Fermentas)). Load the gel with x μl of LD-mixed samples next to the first well with 5 μl marker. The amount loaded for PCR samples depends on the concentration and the size of the products. Note that PCR tubes should be closed to prevent drying up exposed to air.

Running the Gel

When all samples have been loaded, connect the power supply. Ensure that the plus pol is at the opposite site of the loaded wells. Run big gels applying 100 V for a more accurate separation for about 1 h. The time may be adjusted according to the loading front.

Staining the Gel

To make the separated DNA bands visible under the UV light, the samples were stained in a EtBr bath for about 10 min. Then, the gel was rinsed in water for approximately 5 min. The EtBr stained gel was exposed to UV light under a gel documentation station, a gel photo was taken and saved under the >>iGEM2012<< folder. The file should be labelled with the date, group number and short description of what was analyzed. A printed version should be pasted into the group notebook.