? understand how a restriction enzyme works
? analyze a photograph of electrophoresis
? understand how gel electrophoresis separates DNA molecules in a mixture
? how to use electrophoresis to separate DNA fragments
? determine unknown DNA fragment sizes when given DNA fragments of known size
casting tray and comb
crushed ice container
electronic scale with tare
250 ml Erlenmeyer flasks
micropipet and tips
1.5 ml reaction tubes and racks
restriction enzymes (HindIII, EcoRI, BamHI)
10x TEA buffer
37? C water bath
Add 10 ml of 10x TEA buffer and 90 ml of distilled water into a graduated cylinder to create a 1x TEA buffer solution. Add this to the Erlenmeyer flask containing the 0.8 ml of agarose.
Dissolve and boil the agarose solution in a microwave, about 2-3 minutes.
Place clean bottom of the casting tray in place, and pour in the agarose solution. Place the casting comb in place. Allow gel mold to set undisturbed until almost opaque, about 10 minutes.
Fill a graduated cylinder with 50 ml of 10x TEA buffer and 450 ml of distilled water, creating 500 ml of 1x TEA buffer.
In each of the four restriction enzyme tubes, combine 1.0 ?l of restriction buffer, 7.0 ?l of distilled water, 1.0 ?l of the specific enzyme (either HindIII, EcoRI, or BamHI). For the control, add no enzyme. Close the caps tightly and place them evenly balanced in the microcentrifuge and spin for 2-3 seconds. Place the tubes in the 37? C water bath.
When the gel has solidified remover the comb in a careful straight up motion. Remove the glass plate bottom without disturbing the gel and place it in the electrophoresis box with the wells towards the cathode end. Pour the prepared 1X TEA buffer carefully over the gel until the liquid level completely covers the gel and is about 1 or 2 mm above the surface of the gel.
Add 2 ?l of loading dye to each of the enzyme tubes using the micropipet and spin them in the centrifuge. Extract 10 ?l of the first sample and load it into the first well. Repeat this with the other samples, changing tips between each.
Attach the power supply to the electrophoresis box. Set it to 100 volts and 40 milliamps and activate it. After about 45 minutes or until the dye is approximately ? of the down, turn off the power supply and disconnect the box. Using gloves, remove the gels from the box and place them on the transilluminator.
The instructor will carry out the photography of the electrophoresis gel.
Clean the lab area.
IV. Observations and Results
HindIII EcoRI BamHI Control
Distance Act. BP Distance Cal. BP Distance Cal. BP Distance Cal. BP
3.4 cm 25,000* 3.5 cm 23,000 3.8 cm 19,000 3.7 cm 20,000
4.8 9,416 5.3 7,800 4.2 15,000 ———— ————
5.9 6,557 6.4 5,200 5.7 6,800 ———— ————
6.7 4,361 7.1 4,000 5.9 6,500 ———— ————
11.3 2,322 8.7 3,300 6.7 4,300 ———— ————
12.1 2,027 ———– ———— ———— ———— ———— ————
* = rounded base pair
All calculated base pairs (Cal. BP) are rounded figures.
Restriction enzymes are enzymes found in bacteria. These are enzymes that are able to cut through the phosphate-sugar backbone of DNA at restriction sites. Restriction sites are certain base sequences recognized by these enzymes. In bacteria, restriction enzymes act as a defense against invading viruses. When the viral DNA is release into the cell, the restriction enzymes cut it into pieces, rendering it useless and unable to act upon the cell. Any other bacteria entering the cell will also be cut if it contains the base sequence recognized by the enzyme. Every species of bacteria has at least one restriction enzyme. Restriction enzymes are used in genetic engineering to make complementary cuts that allow the insertion of a genetic code into a genome. In electrophoresis, restriction enzymes cut at the restriction sites on the DNA sample. It cuts as many times as the base sequence appears on the sample.
After the sample is cut, buffers, dye, and a substance called ethidium bromide is added to the sample. It is then placed into the well of an agarose gel. An electrical current is run through this, and because DNA has a negative charge it is dragged through it towards the positive end. The DNA weaves through the agarose gel, the smallest pairs going the farthest simply because they are more maneuverable. The longer segments move more slowly through the agarose. When the sample has run about ? of the way through the gel, the current is disconnected, stopping the movement of the DNA. The gel is then placed on an ultraviolet transilluminator. Ethidium bromide is sensitive to UV rays, so it is seen under the transilluminator. A picture is then taken and the distance and base pairs can be measured and calculated.
The buffer used in this is TEA buffer. It is made of Tris and EDTA. Tris keeps the pH constant at about 8.0, and EDTA pulls out low levels of sodium acetate.
Since electrophoresis essentially measures the distance between restriction sites of a certain restriction enzyme, it is helpful in murder and rape cases, where blood or semen of the suspect is found as evidence. In the case of rape, a restriction enzyme is added with the blood or semen evidence. A blood sample is taken from the suspect and DNA is spooled from it. The same restriction enzyme is added to it, and both samples are run through electrophoresis. Since every single person has different genetic material, a match in segments between restriction sites would be an impossibility to be classified pure coincidence. This would clearly identify the suspect as the perpetrator. A difference in segment lengths would clear the suspect, as the DNA would be clearly different.
In our electrophoresis experiment, it is shown how 3 different restriction enzymes act completely differently on the same sample of DNA. This is because each enzyme has a different restriction site it acts upon. The control in this experiment simply shows that DNA without any cuts would run, but would run as a large clump and would run very slowly, as it cannot maneuver easily through the gel matrix.
2. Restriction enzymes are enzymes that use DNA as a substrate. When the proper base sequence, called a restriction or recognition site, is found the enzyme acts by cutting between the backbone two specified bases.
4. The electricity in electrophoresis acts on DNA as a magnet does to another magnet. DNA has a slightly negative charge. The samples containing DNA are loaded at the cathode or negative end. When the power is activated, the DNA is attracted towards the positive end of the electrophoresis box. The agarose gel provides a means of slowing the DNA down. The DNA fragments must work through the gel matrix in order to reach the end.
6. The loading acts as a point of reference. It allows the person performing the experiment to see how far down the DNA sample has moved. The DNA is photographed using ethidium bromide, a UV-sensitive substance and an ultraviolet transilluminator to highlight the DNA strands.