Gel electrophoresis of DNA
In the previous days experiment , we isolated human DNA from cheek cells, and now it is time to analyze the PCR product. To do this we are going to use the charged nature of DNA to force it through a sieving matrix.
The matrix we will employ is called “Agarose” , a carbohydrate derived from kelp and algae.
When agarose is hydrated and heated, it polymerizes into a complex matrix (gel) with gaps between strands.
When DNA is placed into this matrix in the presence of buffer, the negative charge of the phosphate backbone is attracted to the positive pole in an electric field and the DNA must find its way through the matrix. Large molecules encounter more resistance than small molecules and thus a size separation occurs. Depending on the density of the matrix, the overall migration can be approximated by the inverse square of the molecular weight and a matrix concentration of 0.8% can resolve DNA fragments from 400 bp to 20,000 bp.
Since many factors can alter the mobility, a size standard is always used to “calibrate” a particular gel.
The DNA can be added to the gel matrix by being loaded into a well cast into the gel and trapped into the well by being associated with a solution of higher density than the buffer. Typically this is glycerol. A colored dye of known size is added to the DNA sample to estimate how long the electrophoresis should take.
Typically voltages of 100 volts are used for separations and depending on the buffer system and volume, this will yield amperage of 100 – 250 mAmps. Heat generated during the process will dictate how much voltage can be applied to achieve a certain speed. Generally, slower runs give higher resolution.
overview of electrophoresis process.
Protocol:
1) Prepare 50 mls of a 1.05 % agarose gel. 0.525 g agarose/50 mls 1x TAE buffer
2) Melt in microwave. Tape ends of gel cast plate. Pour gel into cast until about 5-6 mm thick. Add well comb.
3) Gel will harden over 20 mins. When gelled completely, remove comb.
4) Cover gel with enough buffer to submerge the gel below 1-2 mm buffer. (1 x TAE buffer)
5) Prepare samples with loading dye by mixing 8-10 l DNA with 3-4 l loading dye on a piece of parafilm.
6) Using a pipetteman, load the sample into the well.
7) Electrophorese for 45 mins to 1.5 hours (see gel, until front dye is 1 cm from end.)
Visualization of DNA
The most sensitive way to visualize DNA is to employ a fluorescent gel stain and excite this with UV light.
We will use ethidium bromide , which intercalates into the DNA bases and fluoresces when excited by 260 nm light.
8)Soak gel in a weak solution of EtBr and water for 10 mins. Destain in water for 10 mins.
9)Visualize on transilluminator light box and take a picture.
Plot distance migrated from well versus molecular size of standard on semilog paper and estimate size of unknown DNA.
Appendix:
Loading dye recipe
Glycerol & bromophenol blue (6x)
3ml glycerol (30%), 25mg bromophenol blue (0.25%) , dH2O to 10mL
Buffer recipe
50 x TAE
- Add the following to 900ml distilled H2O
- 242g Tris base
- 57.1ml Glacial Acetic Acid
- 18.6 g EDTA
- Adjust volume to 1L with additional distilled H2O