Discussion Questions
- Why does DNA move through the gel matrix when electrical current is applied to the gel? It moves through the gel because, since the gel is placed in a box with a negative electrode on one side and positive on the other side and the DNA has a negative molecule charge so when the electronic current passes through the DNA will follow the curremy towards the positive end of the box through the gel.
- What factors affect the rate at which DNA fragments move through the gel? The factors that affect the rate in which the DNA fragments move through the gel are the size of the segments, the longer the segment the slower it will move across the gel.
- Would you expect DNA pieces of a particular size to move faster or slower in a gel with a higher percent of agarose? Explain why. Yes it will depend on their size because, the shorter are faster and the longer are slower through a run-time.
- Describe the steps used to analyze a gel once the electrophoresis is completed. -Once the process is completed and the power is turned off the molecules stop moving and they are stuck where they stopped. The UV light exposes the groups in which the DNA and molecules are of similar size and shape. From the information they contain the scientist can access the relative size and shape of the DNA molecules they contain.
- Why do you think control samples are also usually run? In my opinion control samples are usually run because they might be experimental errors. Containing a sample of gel analysis is very difficult and it is important to obtain the correct information. Containing the correct information is important because if you are trying to find a person of a crime its important to find out who did it. You should always know how to do something lie an experiment before you conduct it.
DNA FINGERPRINT
The first step into making a DNA fingerprint is to pipet the restriction enzymes into the DNA. The enzymes are known to work like "scissors" which can cut through the long DNA molecules, at different locations, where the enzymes cut depend on the code within the DNA and within the enzyme. The lengths of the fragments depend on the person because every persons code is different. The second step was to pipet the agarose gel into a container, the job of the agarose gel is to act as a molecular strainer that will allow small pieces of DNA to move around. After that the DNA was pipeted into the same container used before. When the power button is on in the tray the process of electrophoresis happens which is when molecules are moved within an electric current. The DNA has a negative charge which makes it go to the opposite end which is the positive, the gel acts like a strainer which also allows the smaller strands to move more easily and they move across faster. Once the process of electrophoresis is completed the fragments are distributed in the gel according to their size. When the process is occurring a nylon membrane is put on top of the DNA which looks like a small sheet of paper. After it the probes are added which attach themselves to DNA according to when they encounter a certain sequence of code. The probe that does not attach itself will wash away. Once the x-ray film is exposed to the DNA it exposes corresponding areas on the x-ray film. The next step is putting the x-ray film into the developer which exposes where the probes attached themselves to DNA. From this you receive your DNA fingerprint.
Summary : Their is three tubes which contain the identical DNA fragments, that will be cleaved with different restriction enzymes to yield them into different sizes. Enzyme one cuts the DNA into fragments A and B, enzyme two cleaves the DNA at the fragments of C and D, adding both enzymes yields fragments A, E and D. In the second step it is known that Gel electrophoresis is one of the most useful means of separating DNA fragments. The agarose is molded with well, which is placed in a buffer solution and hooked up to positive and negative electrodes. A well is reserved for the placement of DNA of known sizes and then the power supply is turned on. The blue dye is negatively charged and they migrate to the positive electrode, as does the DNA. The smallest fragments move faster. Once the blue dye is reached to the bottom the power is turned off, a fluorescent dye is used to stain the DNA fragments. When a researcher wants to determine which DNA fragment contains a DNA sequence of interest he creates a blot which means a DNA copy. The gel is then transferred to a salt solution and nylon filter is placed on top. The salt solution attracts the DNA through the gel toward the nylon filter where DNA adheres. The DNA is treated to adhere it permanently and then it is placed with a radioactive probe. The DNA will hybridize to the band of interest. Then a piece of X-ray film is placed over the filter and exposes the film, revealing the locations of hybridization. From this an identical agarose gel can be created and a band interest can of be cut out of the gel.
Discussion Questions
1. After electrophoresing DNA fragments through an agarose gel, you cut out a DNA band that is closest to the positive electrode. Does this band contain DNA fragments that are the smallest or the largest on the gel?
-The smallest because the smallest DNA fragments move faster toward he positive electrode.
2. What happens to the DNA molecules when they are soaked in a basic solution?
-The DNA denatures, separating into single stranded. The basic solution is used in the blotting procedure to separate double-stranded DNA into single stranded.
3. In an experiment, you digest a linear piece of DNA with two different enzymes and then electrophorese the resulting fragments on an agarose gel. You see the following DNA bands on the gel from uncut DNA, DNA cut with restriction enzyme 1, and DNA cut with restriction enzyme 2. How many sites within the original piece of DNA does each enzyme digest the DNA?
-Enzyme 1 cuts at 1 site. Enzyme 2 cuts at 3 sites.
Discussion Questions
1. After electrophoresing DNA fragments through an agarose gel, you cut out a DNA band that is closest to the positive electrode. Does this band contain DNA fragments that are the smallest or the largest on the gel?
-The smallest because the smallest DNA fragments move faster toward he positive electrode.
2. What happens to the DNA molecules when they are soaked in a basic solution?
-The DNA denatures, separating into single stranded. The basic solution is used in the blotting procedure to separate double-stranded DNA into single stranded.
3. In an experiment, you digest a linear piece of DNA with two different enzymes and then electrophorese the resulting fragments on an agarose gel. You see the following DNA bands on the gel from uncut DNA, DNA cut with restriction enzyme 1, and DNA cut with restriction enzyme 2. How many sites within the original piece of DNA does each enzyme digest the DNA?
-Enzyme 1 cuts at 1 site. Enzyme 2 cuts at 3 sites.