BME 310 Lab 11 Electrophoresis, John G. Webster4/10/00

Introduction: Electrophoresis may be defined as the movement of charged particles of a solid phase in response to an electric field placed in a buffer solution (liquid or gel). Depending on the mobility of the particles, different materials migrate through the buffer at different speeds. The mobility of a molecule is its velocity (m/s) divided by the electric field gradient (V/m). Given a particular buffer medium, a particle’s mobility is determined by the net charge of the particle, the size and shape of the particle, and various other physical conditions. If enough time is allowed, the different components of materials are separated in the medium and can be discerned and analyzed. Although the technique was first observed in 1807, electrophoresis is currently used in the clinical laboratory for several purposes. The most important purposes are to measure quantities of the various types of proteins in plasma, urine, and cerebrospinal fluid; to separate enzymes into their component isoenzymes; and to identify antibodies or DNA.

Before the lab: Read material about electrophoresis from J. G. Webster (ed.), Bioinstrumentation, Section 3.15 at the coursepage

Laboratory Equipment:

1Carolina forensics electrophoresis kit

2HP E3630A power supply

3Deionized water

4Beaker

5Balance

61 TBE buffer

7Agarose, 0.8%

At the lab

1When you get a chance, read the background material on pp. 2 to 3 of the lab manual.

2Put comb into gel rig.

3Obtain melted agarose, let cool slightly, and pour into gel rig until dams are reached on each side (about 10 mL of agarose).

4While waiting for the gel to cool (about 30 min), cut two squares 42  22 mm in size out of the carbon sheet. You can also make sure you have tubing on the end of your 1 mL syringe to act as an adapter for the pipette tips.

5Once gel has solidified, carefully remove comb. Pour a little more than 10 mL of TBE into gel rig, making sure that the surface of the gel is just covered and that end reservoirs are full.

6Using the syringe and yellow pipette tips, load the complete volume of each DNA vial into separate wells. Hold the pipette tip just above the well, but under the surface of the TBE solution. Slowly inject the DNA. Be sure to use a new pipette tip for each well.

7Place the alligator clips and carbon sheets in the end reservoirs as shown in the manual. Attach the positive lead to the +20 V output on the dc power supply. Attach the negative lead to the –20 V output on the dc power supply. Turn on power supply and adjust the 20 knob until voltage reads 22.5 V. Record the voltage gradient (V/m) across your gel.

8The gel must now run for approximately 2 h.

9Once the loading dye is almost at the end of the gel (about 1 cm away) turn off voltage supply and remove electrodes. Throw away carbon squares and rinse off alligator clips. Pour the TBE solution in the gel rig into the sink.

10Pour just enough Carolina BLU stain to cover the gel. Let sit for 4 min. Remove stain.

11Fill the rig half full with deionized water. Let sit for about 5 min. Pour off water and repeat again 2 or 3 more times, or until you observe stained DNA bands. Make a sketch of your gel.

Results

1Give the voltage gradient across your gel.

2What is the polarity of the DNA molecules? Specifically, why are they this polarity?

3Provide a sketch of your bands on your gel rig. Who is guilty? Was the evidence contaminated? Justify your results.

4What is the velocity of each band you observed?

5What are the advantages and disadvantages of using a stronger voltage source for running electrophoresis?

6Calculate the mobility of each band you observed. List the factors that affect mobility.

7Give any suggestions for improvement.

References:

  1. McGraw-Hill Encyclopedia of Science and Technology. 7th Edition. 1992, Volume 6, pg.257–260.
  2. Webster, J. G. (ed.). Bioinstrumentation.
  3. Van Nostrand’s Scientific Encyclopedia. 8th Edition. 1995, pg.1131–1133

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