This is a draft of what I think my final experiment simulation will look like:
The DNA sample that is being amplified will be simulated as honey.
DNA primers will be dish soap (blue).
DNA polymerase will be rubbing alcohol or vegetable oil.
Excess nucleotides will be water.
When the student conducts the "experiment," the correct order will end up being the honey, dish soap, water, and then the rubbing alcohol or vegetable oil. The students will then have to list the temperatures and what happens at each temperature, and choose a reasonable number of cylces.
The winner (the group that has the order, the temperatures, the descriptions, and the number of cycles correct) will receive a prize.
Wednesday, February 6, 2013
Final Product?
Right now, I am unsure of what my final product or presentation would be on. I am considering having the class perform a simulation of a PCR, in order to teach the class the basic concepts behind scientific research. PCRs are integral to many types of scientific research, and it would be useful to know how to construct a prediction of the gel, and actually perform a simulation.
My idea for the simulation involves using oil as the polymerase, and different food colourings to indicate the other reagents involved in conducting PCRs.
My idea for the simulation involves using oil as the polymerase, and different food colourings to indicate the other reagents involved in conducting PCRs.
General PCRs: Protocol
1. Retrieve a sample containing a nucleotide sequence (can be from hair, blood, skin, etc. or from a known organism).
2. You will also need DNA primers. These are short single stranded DNA that attach to nucleotide sequences, thus creating a complementary strand of nucleotides.
3. DNA polymerase is another necessary element in a PCR. This enzyme is very sensitive to temperature, so it should only be taken out of the freezer when needed. The enzyme attaches the nucleotides to form a complementary base pair, synthesizing a full complementary nucleotide strand of DNA. Though there are many different types of DNA polymerases, the one most commonly used is Taq polymerase, because it is derived from heat-resistant bacteria, and improves the ability to perform a PCR (which involves many temperature changes). Taq is also a cheaper type of polymerase, so it is a better choice to start with.
4. An excess of nucleotides will also need to be placed into the mixture, so that the polymerase has something to work with. Nucleotides contain Adenine, Thymidine, Cytosine, and Guanine (A, T, C, G).
5. The DNA segment (can be diluted) is placed in a tube containing the items above (be sure to add the Taq polymerase last, so it will be more effective). The tube will then be placed in a thermoregulator, which can easily adjust temperatures. The solution is first heated to at least 94C. This step breaks the hydrogen bonds, thus allowing the strands to separate. This is called denaturation. Then, the tube is cooled to about 54C; at this temperature, the DNA primers start biding to the single stranded DNA. At 72C, the process of DNA polymerisation increases rapidly, creating the double stranded DNA molecules.
6. The cycle can be repeated multiple times depending on the quantity of DNA needed. The most common number of cycles is 30 cycles. Since each cycle doubles the DNA once, 30 cycles results in 2^30 samples.
2. You will also need DNA primers. These are short single stranded DNA that attach to nucleotide sequences, thus creating a complementary strand of nucleotides.
3. DNA polymerase is another necessary element in a PCR. This enzyme is very sensitive to temperature, so it should only be taken out of the freezer when needed. The enzyme attaches the nucleotides to form a complementary base pair, synthesizing a full complementary nucleotide strand of DNA. Though there are many different types of DNA polymerases, the one most commonly used is Taq polymerase, because it is derived from heat-resistant bacteria, and improves the ability to perform a PCR (which involves many temperature changes). Taq is also a cheaper type of polymerase, so it is a better choice to start with.
4. An excess of nucleotides will also need to be placed into the mixture, so that the polymerase has something to work with. Nucleotides contain Adenine, Thymidine, Cytosine, and Guanine (A, T, C, G).
5. The DNA segment (can be diluted) is placed in a tube containing the items above (be sure to add the Taq polymerase last, so it will be more effective). The tube will then be placed in a thermoregulator, which can easily adjust temperatures. The solution is first heated to at least 94C. This step breaks the hydrogen bonds, thus allowing the strands to separate. This is called denaturation. Then, the tube is cooled to about 54C; at this temperature, the DNA primers start biding to the single stranded DNA. At 72C, the process of DNA polymerisation increases rapidly, creating the double stranded DNA molecules.
6. The cycle can be repeated multiple times depending on the quantity of DNA needed. The most common number of cycles is 30 cycles. Since each cycle doubles the DNA once, 30 cycles results in 2^30 samples.
Tuesday, February 5, 2013
The Importance of PCRs
A common technique used in the research lab is the PCR (Polymerase Chain Reaction). It is used to amplify trace amounts of DNA within a liquid. These amplified segments of DNA can then be compared to DNA from a known source, thus letting the researcher know if they have amplified the right gene, or can tell them the source of a certain segment of DNA. PCRs are run in small tubes, and a small portion of the amplified sample can be mixed with blue dye and run on a gel (see previous post on Gel Electrophoresis) for comparison. Often, scientists use a ladder that serves as a comparison point in terms of sizes. The ladder has known sizes of DNA bands spread across a range on the gel.
The picture above shows one of my sample PCRs. In this one, I used different concentrations of plasmid, and got slightly different results (this will be discussed later). From the picture, the DNA ladder can be seen on the left. The very slight band of DNA matching up with the three PCRs after that corresponds to the size of the fragment.
The picture above shows one of my sample PCRs. In this one, I used different concentrations of plasmid, and got slightly different results (this will be discussed later). From the picture, the DNA ladder can be seen on the left. The very slight band of DNA matching up with the three PCRs after that corresponds to the size of the fragment.
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