I. Purpose perform electrophoresis using restriction enzymes and lambda DNA 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 II. Materials agarose powder casting tray and comb camera crushed ice container distilled water DNA samples electronic scale with tare electrophoresis box 250 ml Erlenmeyer flasks film graduated cylinder hood ice loading dye microcentrifuge micropipet and tips 1. 5 ml reaction tubes and racks restriction buffer restriction enzymes (HindIII, EcoRI, BamHI) 10 x TEA buffer UV filter UV transilluminator 37 C water bath weighing boat III.
Procedure Place the weighing boat on the scale and tare. Weigh out 0. 8 ml of agarose powder and place it into a 250 ml Erlenmeyer flask. Add 10 ml of 10 x TEA buffer and 90 ml of distilled water into a graduated cylinder to create a 1 x 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 10 x TEA buffer and 450 ml of distilled water, creating 500 ml of 1 x 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).
The Essay on Electrophoresis And DNA Fingerprinting
... DNA fingerprinting, or electrophoresis, is used to separate out and therefore determine the different sizes of DNA fragments which are cut by restriction enzymes. Since restriction enzymes ... per 100 ml of buffer. The DNA fingerprinting experiment only called for 50ml of buffer, therefore only 0.4 grams ... were unplugged. Carefully, the gel was removed and placed in a UV illuminator. Bands in the gel ...
For th 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 1 X 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 milli amps 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. V. Conclusions Electrophoresis literally means “to carry with electricity.” It is the use of restriction enzymes and electrical current to measure segments of DNA from a sample.
The Essay on DNA Fingerprinting
... how many restriction sites were there in lane three 8.From the gel sketch, which DNA samples appear to have been cut into the ... specific sequence of bases the enzyme is searching for ... doing this lab Background 1.What are restriction enzymes 2.When added to a DNA sample, what do restriction enzymes do 3.What do you call the ...
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.
The Term Paper on Dna And Restriction Enzyme
DNA and Restriction Enzyme Interaction: Identification Of An Unknown Plasmid Abstract The fundamental purpose of these experiments is to research the interaction between DNA molecules and restriction enzymes. Two different experiments are performed. In the first experiment, two restriction enzymes, Hind III and Bam H 1 interact with an unknown plasmid and the resulting DNA fragments are separated ...
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. VI. Questions 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. 3. Restriction enzymes are found naturally in bacteria. They act as a protection against viral infections, as they break down incoming viral DNA. 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. 5.
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..
