Analysis of Plasmid DNA I (Gel Electrophoresis)
Introduction
During the last 2 labs you isolated plasmid DNA. Today you will analyze your DNA sample. One of the most widely used analytical methods in molecular genetics and biochemistry is electrophoresis, primarily because of the ease - and relatively low cost - of separating nucleic acids and proteins by this method. Electrophoretic techniques are extremely versatile and capable of high-resolution separations.
A workhorse method in molecular genetics is agarose gel electrophoresis, with which we will gain experience in this lab. Agarose, which is extracted from seaweed, is a linear polymer that is composed of galactose subunits. Agarose gels can separate DNA fragments from 200 bp to 50 kb. DNA may be detected in gels by using ethidium bromide, which, upon binding to nucleic acids, fluoresces strongly in the visible region when activated by UV light. Photographing the gels under UV light can make a permanent record of the electrophoretic runs. The ethidium method of detection has the advantages of sensitivity (can detect approximately as low as 20 nanograms of DNA), immediate visibility, and lack of interference in subsequent manipulations.
Our immediate application is that of examining the quality of the plasmid DNA prepared in the previous two labs. Plasmids isolated from cells give a complex pattern of bands representing different topoisomers and polymers. These forms do not migrate on gels in a manner strictly consistent with their sizes. DNA can exist in different conformational isomers:
superhelical circular (form I) - supercoiled or closed circular;
nicked circular (form II) - open circular;
linear (form III).
These DNAs migrate through agarose gels at different rates. Under our conditions, closed circular will migrate the fastest, then linear, then open circular.
An important application of gel electrophoresis is the determination of molecular size. Molecules of linear double-stranded DNA migrate through agarose gels at rates that are inversely proportional to the number of base pair pairs (a good introduction to electrophoresis theory is given in D. Freifelder, "Physical Biochemistry," 2nd ed., W.H. Freeman, New York, 1982, Ch. 9).
Migration is also affected by the concentration of agarose.
% of agarose Efficient rate of separation (kb)
0.3% 5-60
0.8% 0.7-8
2.0% 0.1-2
Preparation of gel:
We will use a Mini-Sub (submarine) apparatus for routine procedures in this course. Voltages of 100 VDC or less will generally be used, which, while not considered "high-voltage" are still potentially dangerous and require caution. One must be especially careful where the possibility of grounding might occur, as with water lines and plumbing fixtures. Note also that we will include ethidium bromide at low concentrations in the agarose gel mixtures. Care in handling the gels (WEAR GLOVES!) must be exercised due to the mutagenic nature of ethidium.
1. Prepare the agarose melt (your instructor will assist you in this procedure): weigh the agarose into a clean dry 125-ml flask. A 0.8% gel contains 400 milligrams of agarose per 50 ml of 1X TAE buffer.
2. Place the agarose mixture in the microwave oven at the "high" setting for about 30 seconds. Be sure no aluminum foil or any other metal is ever placed in the microwave oven. Swirl the mixture after heating and look closely to see that the gel fragments are totally melted. Re-heat for an additional five seconds. Do not over-boil!
3. Cool the mixture to about 60 degrees centigrade (the flask should be warm but not hot to the hand). Add 1 μl of ethidium bromide to the solution; swirl to mix thoroughly.
4. Attach the rubber stoppers to each end of the gel tray. Pour the warm agarose into the gel tray - make sure the comb is in the tray and clears the bottom of the gel tray by about 1 mm when in place.
5. The gel will become translucent when it hardens (about 20 minutes). Carefully remove the rubber stoppers from each end of the gel tray and submerge the gel tray into the submarine apparatus. Make sure that the hardened gel is fully submerged in the gel buffer. Carefully remove the comb from the hardened gel!
6. Load the DNA sample (sample = 10 μl of DNA solution + 2 μl of 6X loading dye) into the gel slot by placing the pipet tip directly into the slot and very carefully ejecting the sample. This takes some practice if you are not facile with pipetting! You may want to ask your instructor to load the first sample!
7. Cover the gel box and turn on the voltage as soon as possible after loading to avoid diffusion of the samples. After a few minutes, check the direction of migration of the tracking dye to make sure that current is flowing and that the polarity is right. We will run gels at 100 volts. BE SURE TO TURN OFF THE VOLTAGE FIRST, if you open the box to observe the progress of the run or to terminate the run.
Direction of migration: since DNA is a negative molecule due to the fact that the phosphate groups in DNA are ionized, this molecule will migrate toward the positive electrode (anode).
8. View the results of your run under UV light. Your instructor will assist you in this exercise. Caution: Always protect your eyes from UV light!