Agarose Gel Electrophoresis Protocol

Aim: Qualitative analysis of plasmid and genomic DNA by agarose gel electrophoresis.

Principle:

• Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA, or RNA molecules by size.
• This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis).
• Shorter molecules move faster and migrate farther than longer ones.
• The advantages are that the gel is easily poured, does not denature the samples.
• The samples can also be recovered. Integrity of the nucleic acids can be established.
• Agarose is extracted from a sea weed and is a linear polymer of different form of galactose. Agarose gel can be casted by melting the agarose in the presence of desired buffer until a clear, transparent solution is obtained.
• The melted solution is then poured into a mold and allowed to harden. Upon hardening, agarose forms a matrix, the density of which is determined by concentration of the agarose. When an electric field is applied across the gel, DNA which is negatively charged at natural pH migrate towards the anode.
• The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis is ethidium bromide, usually abbreviated as EtBr.
• It fluoresces under UV light when intercalated into DNA (or RNA).
• It can absorb light at a particular wavelength in UV spectrum and emits at a particular wavelength in the visible spectrum. 
• By running DNA through an EtBr-treated gel and visualizing it with UV light, distinct bands of DNA become visible. EtBr is a known carcinogen and should be handled carefully.

Requirements:

1x TAE buffer: 0.04M Tris-acetate, 0.001M EDTA,

Agarose gel(0.8%): Agarose- 0.8 gm,

Ethidium bromide:10mg EtBr powder in 1ml double distilled water,

Gel loading dye: 0.25% Bromophenol Blue, 0.25% Xylene Cyanol FF, 30% Glycerol in water  (store at   4 º C).

Procedure:

Step 1:

1. Seal the edges of a clean dry gel casting tray with tape so as to form a mold. Position the combs 0.5-0.1mm above the plate , so the complete well is formed when agarose is added. Place two combs with 6 wells so as to get 12 wells in a single gel.
2. Prepare sufficient electrophoresis buffer(1x TAE) to fill the electrophoresis tank and prepare the gel.
3. Prepare 0.8% of the agarose gel by dissolving 0.4gm of agarose in 50ml of 1x TAE buffer by heating at a temperature of 60º C and add 2 µl ethidium to the gel to get the final concentration of 0.5 µg/ml.
4. Pour the gel in the gel casting unit with combs. After complete setting the gel, remove the combs and the tapes and mount the gel in an electrophoresis tank. Add enough electrophoresis buffer to cover the gel to a depth of about 1mm. 

Step 2: 

1. Take 15µl of the DNA on a parafilm and add 2.5 ml of the gel loading dye and mixed properly. Gel loading buffer serves 3 purposes. It increases the density of the samples ensuring that the DNA drops evenly into the well. It gives color to the samples thereby simplifying the process. They contain the dyes that move towards the anode at predictable rate in an electric field. Bromophenol blue and  xylene cyanol are 2 dyes that are present in the loading dye. Bromophenol blue migrates to the through the agarose gel approx 2.2 fold faster the same speed as the DNA of 500 bp on a 1% gel and xylene cyanol migrates at the same speed as that of 4kb linear stranded DNA.
2. With the help of a micropipette, load the samples into the well. Close the lid and connect the electric leads and carry out electrophoresis.
3. After electrophoresis for 1:00 to 1:30 hours, turned off the electric current and disconnect the leads. Open the lid and observe the gel on a UV Transilluminator.

Result:

Visualize the distinct DNA bands of Genomic DNA and Plasmid DNA after 0.8% Agarose gel electrophoresis under UV transilluminator.

Choose a site Web Design / HTML Front Page Beginning HTML JavaScript Help