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C. Estrella Bio. 4 Lab Handout: Gel Electrophoresis of DNA Samples Nucleic acid samples are often subjected to gel electrophoresis to characterize the size and number of different fragments in the sample. In this exercise, you will be using different DNA samples, some of which have been digested with restriction enzymes (bacterial enzymes capable of cutting the DNA molecule at specific sites). These samples are then mixed with a tracking dye so you can keep track of their migration since you can't really see the DNA molecules until they have been further processed. The samples are subjected to agarose gel electrophoresis (electrical current is passed through a buffer system and gel). When charged molecules are placed in an electrical field, the molecules will migrate towards one of the electrodes, depending on the net charge of the molecule. Nucleic acids are negatively charged so they will migrate toward the positive electrode. Separation of nucleic acid molecules of different size or conformation is achieved by forcing the molecules to migrated through a support matrix (in this case: agarose gel) in the electrical field. This matrix acts like a sieve to allow small molecules to migrate faster than larger molecules. The result is that molecules separate in the matrix according to their relative size and shape. This exercise will use gel electrophoresis to examine the fragments present in several DNA samples with and without enzyme digestion. In this way you will gain experience in pouring gels, loading DNA samples, and visualizing DNA bands on the gels. Due to the very small volumes of samples to be applied to the gel, samples are routinely handled with a manual micropipet that uses a disposable plastic tip to handle a 10 l to 20 l sample. Materials needed at each group station: one gel electrophoresis box with electrode cords one gel former with comb (to form wells in gel) power supply box flask of agarose in TBE buffer (1X) eight tubes of DNA samples (see next page) micropipet (2 l to 10 l) with disposable plastic tips (yellow) one insulin syringe (1 ml; each smallest line = 0.01 ml or 10 l)

Pick one each of the following DNA samples in small (0.5 ml) tubes:

1. 2. 3. 4. 5. 6. 7. 8. Procedure:

pUC19 plasmid, not digested pUC19 plasmid digested with the restriction enzyme EcoRI pUC19 recombinant, not digested pUC19 recombinant plasmid digested with the enzyme EcoRI Bacteriophage lambda DNA digested with the enzyme HindIII Bacteriophage lambda digested with EcoRI E. coli bacterial DNA digested with EcoRI Human DNA digested with EcoRI

1. Heat the flask of buffer and agarose in the microwave until it starts to boil. Take it out and swirl gently. Make sure you use the heat protecting glove at the microwave station because the flask will be HOT. Repeat this procedure until all the agarose is melted and there are no little specks in the melted agar. 2. Cool the flask of agar until it is comfortable to touch (55 oC). Add 2 l of ethidium bromide to the agarose and mix thoroughly. This is the stain to detect the DNA. Ethidium bromide is a mutagen (comparable to a pack of cigarettes) and solutions containing ethidium bromide should not be handled with bare hands. 3. Set the comb in the groove at the black end of your gel tray. Make a border using masking tape on the open ends of the gel try. Pour the melted agar into the tray until it comes up about a little less than 1/3 the length of a comb tooth (approximately 3 mm). Allow the gel to cool (it will turn opaque when solidified). 4. When cool, take a small amount of buffer and pour it on the gel as a lubricant. Remove the comb straight up and you will be left with tiny wells where the teeth were. Remove the masking tape. Do not tip the gel tray because the gel may "slip" off and be ruined. 5. Place the gel tray between the two posts in the electrophoresis box with the black tape side on the same side as the black dot on the back of the box. Your wells on the gel should also be on this side (negative side). 6. Pour buffer in the box until it just barely covers the gel.

7. Use a manual pipettor with a disposable plastic tip to load 10 l of each DNA sample in each well in the gel. Load the samples in the following order and amount:

Lane 1 2 3 4 5 6 7 8 9 10

Tube # 1 2 3 4 5 6 5 6 7 8

Amount 10 l 10 l 10 l 10 l 3 l 3 l 10 l 10 l 10 l 10

8. Close the box by lining up the electrode posts in the back of the lid with the back of the box and sliding the cover toward you as you hold the box. Make sure the black lead (wire) is plugged in the negative hole and the red lead is plugged into the positive receptacle on the power supply box. 9. Turn on the power supply switch on the power box and adjust the voltage to 100-120 volts. The channel switch lets you read the voltage for either gel box depending on the location of the switch. Make sure the position of the switch matches your gel box when adjusting the voltage. Since each power box can accommodate two gel boxes, two groups will use the same power supply box. DO NOT OPEN THE BOX WHILE THE POWER SUPPLY IS ATTACHED AND ON-----HIGH VOLTAGE----We want this to be an electrophoresis lab, not an electrocution lab!!! 10. Each DNA sample has already been mixed with a blue loading dye which allows you to watch the migration through the gel. When the furthest band of blue is about 3/4 of the way to the end of the gel, turn off the power supply and you are ready to view the sample under the UV transilluminator (about 45 minutes at 120 volts). DO NOT LOOK DIRECTLY AT THE UV LIGHT! UV light causes skin burns, is a mutagen, and will cause severe headaches from eye damage with direct exposure. Always operate with the protective shield in the down position. A photograph can now be taken for a permanent record. (you may have to run the gel a few minutes longer if it appears that the ethidium bromide stain is too dark in the gel making it hard to visualize the DNA banding).

***Draw the bands of each DNA sample now so you will have it available for your lab report. Use the box below with the labeled lanes to record your results.

10 9 8 7 6 5 4 3 2 1 Several points are illustrated with this demonstration: 1. Sample #1 is an uncut circular DNA that will give about 3 bands (depending on the shape that circle takes - single, supercoiled, or dimer). Sample #2 is the same DNA cut with an enzyme to give a single linear DNA molecule. The enzyme can digest the circular DNA t a linear DNA and alter the mobility of the DNA by changing conformation or shape without changing size. The same point is illustrated with sample #3 (undigested) and sample #4 (digested). 2. Sample #4 contains an additional DNA band relative to sample #3. Sample #4 is a recombinant plasmid like that used in the recombinant DNA demonstration last week and you can now see the extra DNA fragment present in the recombinant DNA! 3. Samples #5 and #6 are loaded in two amounts because there are both very large and very small DNA fragments in the samples. Note that a specific pattern of bands is present when the bacteriophage DNA is digested with HindIII (sample #5) and a different pattern of bands is apparent after digestion with EcoRI (sample #6). By comparing sample #6 with sample #2, it is apparent that digestion of different DNA samples with the same enzyme gives different bands. By comparing sample #5 with sample #6, it is apparent that digestion of the same DNA sample with different enzymes also gives different bands.

4. Compare lanes #2, #4, #6, #8, #9, and #10. Note that the number of DNA bands increases as the size of the genome examined increases. The plasmid pUC19 is about 3,000 bases long and has one EcoRI site, the recombinant plasmid pUC19 is 5,000

bases and has two EcoRI sites, bacteriophage lambda is 50,000 bases long and has five EcoRI sites, E. coli DNA is about 10 6 bases and has hundreds of EcoRI sites, and human DNA is about 3x109 bases with thousands of EcoRI sites. As the size of the genome increases, the number of DNA fragments generated by a particular restriction enzyme also increases.