Separating Fragments of DNA by Gel Electrophoresis
There are three tubes that contain identical DNA fragments. Each one will be cleaved with different restriction enzymes to yield the fragments in different sizes. The first enzyme cuts the DNA into fragments A and B and the second enzyme cleaves the DNA at the fragments of C and D. Adding these two enzymes yields fragments A, E, and D. Gel electrophoresis is one of the most useful step in separating DNA fragments. The agarose is molded with well, placed in a buffer solution and hooked up to positive and negative electrodes. A well is closed for the placement of DNA of identified sizes. Now, the power supply is turned on. The blue dyes charge is a negative that travels to the positive electrode. When the blue dye reaches the bottom of the gel, the power is turned off. To strain the DNA fragments, you use a fluorescent dye. When a researcher creates a blot, or a DNA copy, he/she wants to determine which DNA fragment contains a DNA sequence of interest. For starters, the gel is soaked in a basic solution. This will make the double-stranded DNA denatures into single strands. The gel is transferred to a salt solution and nylon filter is placed on top of the gel. The DNA is adheres because of the salt solution brings the DNA through the gel toward the nylon filter. The filter is treated to adhere it for good. Now, the filter is placed with a radioactive probe. The DNA is complementary and will hybridize to the band of interest. The x-ray film is placed over the filter. The radioactive begins to expose, revealing the locations of hybridization. For further analysis, the DNA in this band can then be manipulated.
Quiz 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 gel because the smallest DNA fragments migrate the fastest toward the positive electrode.
2. What happens to the DNA molecules when they are soaked in a basic solution?
The DNA denatures, separating into single strands because the basic solution is used in the blotting procedure to separate double-stranded DNA into single strands.
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.
The smallest gel because the smallest DNA fragments migrate the fastest toward the positive electrode.
2. What happens to the DNA molecules when they are soaked in a basic solution?
The DNA denatures, separating into single strands because the basic solution is used in the blotting procedure to separate double-stranded DNA into single strands.
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.
Gel Electrophoresis and Gel Analysis
DISCUSSION Questions
1. Why does DNA move through the gel matrix when electrical current is applied to the gel?
DNA moved through the gel matrix when the gel is placed in a box with a negative electrode on one side and a positive on the other, the electric current passes through the DNA. The DNA will follow the current towards the positive end of the box passing through the gel because it has a negative molecule charge.
2. What factors affect the rate at which DNA fragments move through the gel?
Factors affect the rate at which DNA fragments move through the gel are the size of the segments. The longer the segment is, the slower it will move across the gel.
3. 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, because it depends on the size. The shorter it is, the fater it goes and the longer it is, the slower it goes through a run-time.
4. Describe the steps used to analyze a gel once the electrophoresis is completed.
a.) The power is turned off, the molecules stop moving, and they become stuck once they stop
b.) The UV light exposes the groups in which DNA and molecules are of similar size/shape
c.) With the information contained, the scientist can access the relative size/shape of the DNA molecules they have
5. The DNA ladder provides a reference for the size of the DNA. Why do you think control samples are also usually run?
The control samples are mainly run because they might be experimental errors. When containing a sample of gel analysis, it is very difficult, but it is important to keep the correct information. When containing the correct information, it is very important. For example, if you are trying to find the suspect of a crime, you need to make sure it's the correct person. You always need to know the correct information before doing so.
DNA moved through the gel matrix when the gel is placed in a box with a negative electrode on one side and a positive on the other, the electric current passes through the DNA. The DNA will follow the current towards the positive end of the box passing through the gel because it has a negative molecule charge.
2. What factors affect the rate at which DNA fragments move through the gel?
Factors affect the rate at which DNA fragments move through the gel are the size of the segments. The longer the segment is, the slower it will move across the gel.
3. 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, because it depends on the size. The shorter it is, the fater it goes and the longer it is, the slower it goes through a run-time.
4. Describe the steps used to analyze a gel once the electrophoresis is completed.
a.) The power is turned off, the molecules stop moving, and they become stuck once they stop
b.) The UV light exposes the groups in which DNA and molecules are of similar size/shape
c.) With the information contained, the scientist can access the relative size/shape of the DNA molecules they have
5. The DNA ladder provides a reference for the size of the DNA. Why do you think control samples are also usually run?
The control samples are mainly run because they might be experimental errors. When containing a sample of gel analysis, it is very difficult, but it is important to keep the correct information. When containing the correct information, it is very important. For example, if you are trying to find the suspect of a crime, you need to make sure it's the correct person. You always need to know the correct information before doing so.
Gel Electrophoresis is a process that is used to divide parts of DNA into their size/shape. Solutions that contain plasmids of matching size and sequence can result in different bands on an agarose gel. Two basis change how quickly a part of DNA moves through agarose gel. If the part of DNA is more larger, the slower it moves and the more compact the fragment, the faster it will move. This happens because plasmids can take on different shapes depending on how they are coiled. When Gel Electrophoresis is completed, the next step would be to place the gel over an ultra violet light source to uncover the position of the DNA samples. The bands end up glowing because during the process the DNA has been stained with afflidiom bromide.
Create a DNA Fingerprint
To create a DNA fingerprint you first have to pour restriction enzymes into DNA. Restriction enzymes cuts the long DNA molecules at different locations. The location of the cut depends on the code within the DNA molecule and the code within the enzymes. Second, is to pour agarose gel into the tray. It acts like a molecular strainer, allowing smaller pieces of DNA that will mover through easily than larger pieces. Third, pour DNA into the try. The DNA fragment lies within the hole of the agarose gel. Forth, push the "POWER" button on the tray to begin electrophoresis. The DNA fragments have a negative charge, so they move to the trays positive end. Fifth, place nylon membrane on top of the gel. The DNA is transferred to the nylon membrane. Sixth, add probes to the nylon membrane in the tray. The probes attach themselves to the DNA fragments on the nylon membrane. Seventh, place x-ray film on top of nylon membrane in tray. The radioactively are now seen on some of the nylon membrane. Lastly, develop film by dragging it to the developer. Now, you can identify your suspect.