Wednesday, April 10, 2013
Essay/ Final Product
In my presentation, I will first explain different safety measures that need to be taken for scientific experimentation, and then I will explain the principles of PCRs. The interactive activity will be what was described two posts ago.
Research Essay
Since my topic is somewhat broad and covers the importance of research and basic scientific research strategies, I will need to narrow down my topic for my research essay. I think I will focus on the importance of research, and maybe use the basic research strategies as examples of how anyone can conduct research. I guess the purpose of my paper will be to show the non-scientific community that anyone can conduct the basic principles of scientific research, even if they are not experienced in the field.
Presentation
My presentation will focus on teaching the class how to perform a PCR correctly, and what precautions should be taken.
Safety: the students will have to identify the safety equipment that may be needed. Gloves are a necessity, because contamination can be a problem. Bonus points will be awarded for any student that includes the use of an "Ice bucket" in order to keep the enzymes close to their normal temperature.
The DNA sample that is being amplified will be simulated as honey.
DNA primers will be dish soap (blue).
DNA polymerase will be 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 PCR will be conducted in clear cups. The students will then have to list the temperatures and what happens at each temperature, and choose a reasonable number of cylces (about 30).
The winner (the group that has the order, the temperatures, the descriptions, and the number of cycles correct) will receive a prize.
Safety: the students will have to identify the safety equipment that may be needed. Gloves are a necessity, because contamination can be a problem. Bonus points will be awarded for any student that includes the use of an "Ice bucket" in order to keep the enzymes close to their normal temperature.
The DNA sample that is being amplified will be simulated as honey.
DNA primers will be dish soap (blue).
DNA polymerase will be 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 PCR will be conducted in clear cups. The students will then have to list the temperatures and what happens at each temperature, and choose a reasonable number of cylces (about 30).
The winner (the group that has the order, the temperatures, the descriptions, and the number of cycles correct) will receive a prize.
Beer's Law
Beer's Law has to do with spectrophotometers, which were discussed in the previous post. Beer's Law is given by the equation A=ebc, where A is the absorbance (A has no units), E is the molar absorptivity with the units L/(mol*cm), b is the path length (cm), and c is the concentration of the solution (mol/L).
This equation can be used not only to find the absorbance of a solution given the other three variables, but it can be applied to spectrophotemtry. The spectrophotometer could output the absorbance, and b (measure the size of the cuvette) and E (use a graph) are known, so the concentration of the solution could be found.
Beer's Law is useful in not only finding concentrations of solutions one creates. Researcher's apply Beer's Law to their research. In many cases, Beer's Law can be used to determine the concentration of plasmid, DNA, or protein in a solution, which can then be used for calculations to find out how much would need to be placed in a PCR or other experimental procedure.
This equation can be used not only to find the absorbance of a solution given the other three variables, but it can be applied to spectrophotemtry. The spectrophotometer could output the absorbance, and b (measure the size of the cuvette) and E (use a graph) are known, so the concentration of the solution could be found.
Beer's Law is useful in not only finding concentrations of solutions one creates. Researcher's apply Beer's Law to their research. In many cases, Beer's Law can be used to determine the concentration of plasmid, DNA, or protein in a solution, which can then be used for calculations to find out how much would need to be placed in a PCR or other experimental procedure.
Spectrophotometers
Spectrophotometers are used to measure the concentration of a solution.
A solution is placed in a cuvette (picture below)- about 3/4 of the cuvette is filled, and a beam of light (from inside the spectrophotometer-the cuvette is placed in the open door) is shone through the cuvette. The purpose of the beam of light is to see how much light "gets through" the solution, thus indicating the concentration of the solution.
If a solution is more concentrated, then less light will pass through, because a certain amount of light is absorbed by the solution.
The light is beamed towards the sample solution using an entrance slit, which directs the light. The light will pass through the solution and then reach a source. The source usually measures the wavelength of the light in order to convey how much light passes through.
The number given can either be absorption (the amount of light that does not pass through) or transmission (the amount of light that passes through the solution).
A solution is placed in a cuvette (picture below)- about 3/4 of the cuvette is filled, and a beam of light (from inside the spectrophotometer-the cuvette is placed in the open door) is shone through the cuvette. The purpose of the beam of light is to see how much light "gets through" the solution, thus indicating the concentration of the solution.
If a solution is more concentrated, then less light will pass through, because a certain amount of light is absorbed by the solution.
The light is beamed towards the sample solution using an entrance slit, which directs the light. The light will pass through the solution and then reach a source. The source usually measures the wavelength of the light in order to convey how much light passes through.
The number given can either be absorption (the amount of light that does not pass through) or transmission (the amount of light that passes through the solution).
Wednesday, February 6, 2013
Expansion on Presentation Ideas
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.
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.
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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