AGAROSE GEL ELECTROPHORESIS

AGAROSE GEL ELECTROPHORESIS

AIM:

To demonstrate Agarose gel electrophoresis in the microbiology laboratory

PRINCIPLE:

Agarose gel electrophoresis is a routinely used method for separating proteins, DNA or RNA. Nucleic acid molecules are size separated by the aid of an electric field where negatively charged molecules migrate toward anode (positive) pole. The migration flow is determined solely by the molecular weight where small weight molecules migrate faster than larger ones. In addition to size separation, nucleic acid fractionation using agarose gel electrophoresis can be an initial step for further purification of a band of interest. Extension of the technique includes excising the desired “band” from a stained gel viewed with a UV transilluminator. In order to visualize nucleic acid molecules in agarose gels, ethidium bromide is commonly used dye. Illumination of the agarose gels with 300-nm UV light is subsequently used for visualizing the stained nucleic acids. Furthermore, samples can be recovered and extracted from the gels easily for further studies. Another advantage is that the resulting gel could be stored in a plastic bag and refrigerated after the experiment. Depending on buffer during electrophoresis in order to generate a suitable electric current and to reduce the heat generated by electric current can be considered as limitations of electrophoretic techniques.

Materials Required:

Buffers and Solutions:

Agarose solutions, Ethidium bromide, Electrophoresis buffer.

Nucleic Acids and Oligonucleotides: DNA samples & DNA Ladders.

·         An electrophoresis chamber and power supply.

·         Gel casting trays, Sample combs, Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).

·         Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.

·         Ethidium bromide, a fluorescent dye used for staining nucleic acids.

·         Transilluminator (an ultraviolet light box), which is used to visualize ethidium bromide-stained DNA in gels.

 BUFFER AND AGAROSE GEL PREPARATION:

1.       Prepare a 50x stock solution of TAE buffer in 1000m of distilled H₂O:

For this weigh 242 g of Tris base in a chemical balance. Transfer this to a 1000ml beaker.
Prepare EDTA solution (pH 8.0, 0.5M) by weighing 9.31g of EDTA and dissolve it in 40ml distilled water. EDTA is insoluble and it can be made soluble by adding sodium hydroxide pellets. Check the pH using pH meter. Make the solution 100ml by adding distilled water.
Pipette out 57.1 ml of glacial acetic acid.Mix the Tris base, EDTA solution and glacial acetic acid and add distilled water to make the volume to 1000ml

2.       Prepare sufficient electrophoresis buffer (usually 1x TAE ) to fill the electrophoresis tank and to cast the gel:

 For this we take 2ml of TAE stock solution in an Erlenmeyer flask and make the volume to 100ml by adding 98ml of distilled water. The 1x working solution is 40 mM Tris-acetate/1 mM EDTA

3.      Prepare a solution of agarose in electrophoresis buffer at an appropriate concentration:

 For this usually 2 grams of agarose is added to 100ml of electrophoresis buffer.

Agarose Concentration in Gel (% [w/v])	Range of Separation of Linear DNA Molecules (kb)
0.3	5-60
0.6	1-20
0.7	0.8-10
0.9	0.5-7
1.2	0.4-6
1.5	0-2-3
2.0	0.1-2

4.      Loosely plug the neck of the Erlenmeyer flask. Heat the slurry in a boiling-water bath or a microwave oven until the agarose dissolves.

5.      When the molten gel has cooled, add 0.5µg/ml of ethidium bromide. Mix the gel solution thoroughly by gentle swirling. 

6.      While the agarose solution is cooling, choose an appropriate comb for forming the sample slots in the gel.

7.      Pour the warm agarose solution into the mold and the gel should be between 3 - 5 mm thick. 

8.       Allow the gel to set completely, then pour a small amount of electrophoresis buffer on the top of the gel, and carefully remove the comb. Pour off the electrophoresis buffer. Mount the gel in the electrophoresis tank.

9.      Add just enough electrophoresis buffers to cover the gel to a depth of approx. 1mm.

10.  Mix the samples of DNA with 0.20 volumes of the desired 6x gel-loading buffer.

11.  Slowly load the sample mixture into the slots of the submerged gel using a disposable micropipette or an automatic micropipettor or a drawn-out Pasteur pipette or a glass capillary tube. Load size standards into slots on both the right and left sides of the gel.

12.  Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive anode (red lead). Apply a voltage of 1-5 V/cm.

13.  Run the gel until the bromophenol blue have migrated an appropriate distance through the gel.

14.   The gel tray may be removed and placed directly on a transilluminator. When the UV is switched on we can see orange bands of DNA. 

CONGLUSION:

Schematic illustration of a typical horizontal gel electrophoresis setup for the separation of nucleic acids.

Nucleic acids running on an electrophoresis can be detected by staining with a dye and visualized under 300-nm UV light. Staining and visualization of DNA are conducted by using either ethidium bromide. Ethidium bromide can be used to detect both single- and double-stranded nucleic acids (both DNA and RNA). In fact, most fluorescence associated with staining single-stranded DNA or RNA is attributable to binding of the dye to short intrastrand duplexes in the molecules. The banding pattern of DNA resolved through the gel by recorded images. Images of ethidium bromide stained gels may be captured by using transmitted or incident UV light.

However, the amount of nicking of the DNA is much lower at 302 nm compared to 254 nm. If SYBR Green used instead of ethidium bromide another 10-20-fold increase in the sensitivity using conventional image taking techniques is in the range of possibility. Detection of DNAs stained with this dye requires the use of a yellow or green gelatin or cellophane filter with the camera along with the illumination with 300-nm UV light.

Gel electrophoresis based image analysis. Agarose gels, stained by Ethidium bromide under the UV illuminator