Nucleic acids of  a given type may be separated by polyacrylamide gel electrophoresis because of their electrophoretic mobilities in such gels vary inversely with their molecular masses. However, DNAs of more than a few thousand base pairs cannot penetrate even a weakly cross-linked polyacrylamide gel. This difficulty is partially overcome through the use of agarose gels. By using gels with an approximately low agarose  constant, relatively large DNAs in various sizes ranges may be fractionated. In this manner, plasmids (small, autonomously replicating DNA molecules that occur in bacteria and yeast), for example, may be separated from the larger chromosomal DNA of bacteria.
 

Reagents and Supplies

From the locker and store room:
 Buffer Solutions
      50x TAE                   1x TAE:( 0.04M Tris Acetate, 1mM EDTA)pH 8.0
      242 g Tris base
      57.1 mL glacial acetic acid
      10 ml of 0.5 M EDTA, pH 8.0
      distilled water

      10x TBE                    1x TBE 0.089M Tris Borate, 25mM EDTA) pH 8.3
      108 g Tris base
      55 g boric acid
      distilled water
      Loading buffer:         0.25% bromophenol }
                                         Blue                             }           in 1x TAE or TBE
                                         0.2M EDTA                }
                                         50% glycerol              }

      Test Samples
               DNA solution from experiment: Extraction of DNA from Calf or Hog Thymus
               RNA stock solution (0.1 mg/ml)
               Molecular weight marker (1Kbp)

Things to Bring:
      Disposable surgical gloves
      Plastic dish lined with filer paper

Equipment:
     Water bath
     UV lamp
 

Procedure:

A. Gel Preparation
1. Weigh out agaroset make a 15 ml 0.8% suspension in TBE. Place the agarose in a 125 ml Erlenmeyer’s flask and add 1 ml of cold TBE or TAE buffer.

2. Heat the contents of the flask while stirring on a magnetic hot plate. Allow the contents of the flask to reach near boiling. Alternatively, you can heat the suspension in a microwave oven. Replenish volume through lost evaporation by adding hot distilled water.

3. When all the agarose has dissolved in the hot buffer, transfer the Erlenmeyer’s flask into a 50oC water bath. Incubate for at least 10 minutes at 50oC.

4. Swirl the contents of the flask slowly (Avoid bubbles!) to mix thouroughly. Return flask to 50oC water bath.

5. Set up the gel caster and set the comb in the proper position. Gently pour the agarose gel into the gel caster, ensuring that no bubbles are formed.

6. Allow the gel to settle for at least 20 minutes. The gel should look opalescent.

7. Pipette a few ml of the buffer to wet the surface of the set gel. Allow the buffer to run between the teeth of the comb, and gently lift the comb to separate it from the gel.The comb will leave uniform wells in the gel.

8. Transfer the gel into the electrophoresis tank. Fill the tank with enough buffer (TBE or TAE) such that the gel is submerged in about 1.5 mm buffer. If using ethidium bromide to visualize nucleic acids, add enough ethidium bromide stock solution to a concentration of 0.5g/ml running buffer.

If using ethidium bromide to visualize the nuleic acid bands, add enough ethidium bromide stock to a concentration of about 0.5g/ml.

In case bubbles are formed, quikly aspirate off the bubbles using a pasteur pipette, or push the bubbles to the side
 

B. Sample Preparation
1. Pipette 5ul of loading buffer to a spot plate.

2. To this buffer add the appropriate volume of DNA solutions as calculated.

3. Mix gently and load into one of the wells of the gel.

4. Repeat procedure 2 for RNA and for the MW markers.

5. Turn on the power supply and run the gel at 50 V until the dye in the loading buffer has migrated about ¾ of the length of the gel.

6. Turn off the power supply and transfer the gel into a plastic dish layered with filter paper.

7. Visualize the bands under UV light. The bands corresponding to the nucleic acids will be stained bright fluorescent orange.

8. Measure the migration distance of each band gel.

Calculate the appropriate volume required to give 2g of each of the following samples

PRECAUTION: Use gloves in handling the agarose gels with ethidium bromide. Dispose of the excess staining solution in the designated jar. In case of contamination, remove contaminated material and splash with 70% ethanol.
 

C. Estimation of Relative Molecular Weights of Loaded Samples
1. Plot migration distance versus the logarithm of the molecular weight (or the number of base pairs), using data from the molecular weight standard lane.

2. Estimate the relative molecular weight(or number of base pairs) of DNA and RNA samples.

Note: As safer but more time consuming alternative way for visualizing nucleic acids run in agarose, the gel may be stained with methylene blue. Stain the gel in 0.02% methylene blue solution in 1omM Tris acetate, pH 8.3 for 1-2 hours at 4oC. Avoid excessive exposure of the gel to sunlight. After staining,wash off excess stain with several changes of distilled water for 5-8 hours, or until the bands(blue on pale blue background) become visible.The detection limit for the methylene blue-stained gel is 250ng of nucleic acid/cm band. For single stranded DNA or RNA, ethidium bromide’s detection limit is 5-10 times lower.
 
 
 

Links :
Back to Main Page
Chemistry 145.1
Human Genome Project and Bioinformatics
Experiment No. 1
Experiment No. 2
Experiment No. 3
Experiment No. 4
Experiment No. 5
Experiment No. 6
Experiment No. 7
Experiment No. 8
Experiment No. 9
Experiment No. 10
Experiment No. 11
Experiment No. 12
References