BME100 f2013:W1200 Group2 L4: Difference between revisions
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'''Thermal Cycling''' | '''Thermal Cycling''' | ||
Thermal cycling is the process of cycling a PCR tube alternatively in cold or hot temperatures in a matter of different specific times. Thermal cycling is of six steps in which includes the initial step, denature, anneal, extend, the final step, and the final hold. In the initial step, the PCR reaction is first exposed to a high temperature of 95 degrees celsius for 3 minutes. Taq polymerase is activated and begins to read the genetic data from DNA, proofreads, and copies the data or target sequence which it will eventually generate. In the second step denature, due to the fact that DNA is composed mostly of proteins, in high heat of 95 degrees celsius for 30 seconds, proteins denature and start losing their tertiary structure which is the two-stranded structure. The two-stranded structure begins to uncoil and separates into two single-stranded structures of DNA. | Thermal cycling is the process of cycling a PCR tube alternatively in cold or hot temperatures in a matter of different specific times. Thermal cycling is of six steps in which includes the initial step, denature, anneal, extend, the final step, and the final hold. In the initial step, the PCR reaction is first exposed to a high temperature of 95 degrees celsius for 3 minutes. Taq polymerase is activated and begins to read the genetic data from DNA, proofreads, and copies the data or target sequence which it will eventually generate. In the second step denature, due to the fact that DNA is composed mostly of proteins, in high heat of 95 degrees celsius for 30 seconds, proteins denature and start losing their tertiary structure which is the two-stranded structure. The two-stranded structure begins to uncoil and separates into two single-stranded structures of DNA. The next step called anneal is the process in which the contents of the PCR tube is exposed to a temperature of 57 degree celsius for 30 seconds. During this process, and with the single strands, primers begin to attach opposite ends of a strand. The 3' primer attaches to the end of strand that contains the hydroxide group, while the 5' primer attaches to the end with the phosphate group. In the fourth step called extend, the process lasts for 30 seconds and exposed to 72 degrees celsius. The taq polymerase is again activated and searches for primers that are attached to existing strands of nucleotides. Once it finds primers, it will attach new units of nucelotides to the 3' end of the parent strand. Polymerase will not add new nucleotides to the 5' end primer and so the new growing strand will extend from one direction only. | ||
''Initial Step'' | ''Initial Step'' |
Revision as of 11:33, 30 October 2013
BME 100 Fall 2013 | Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help | |||||||||||||
OUR TEAMLAB 1 WRITE-UPThe Original Design Part 3 )When we unplugged the wires, coming from the screen, from the "brains/motherboard", the machine's screen went blank.
ProtocolsThermal Cycler Program
DNA Sample Set-up Procedure Materials: Procedure: Positive control: cancer Negative control: Not cancer Step 3: Add DNA/primer mix to positive control.
PCR Reaction Mix -Taq DNA Polmerase
The primer is made up of nucleic acids that serve as a starting point for DNA synthesis.
Research and DevelopmentPCR - The Underlying Technology PCR Reaction Components The components of the PCR Reaction include the template DNA, primers, Taq Polymerase, Magnesium Chloride (MgCl2), and Deoxyribonucleotides (dNTP's). Each component or reagent is added to the PCR tube before running them through the PCR device and software called "Open PCR". The template DNA is the source of genetic information in which the enzyme taq polymerase copies the target sequence from. The Taq Polymerase is an enzyme that can stand high temperatures, making it a thermostable enzyme that is ideal for use in the PCR reaction process. It reads the genetic information from the DNA template, proofreads, copies, and synthesizes new nucleotides to form a new strand that will pair with the 'parent' strand. Primers are pieces or fragments of DNA that attach to the a single-strand of DNA. There are two primers, the 3' primer and the 5' primer which both separately attach to an opposite end of a single strand. With out the primers, the taq polymerase cannot start adding nucleotides to the existing strand. Magnesium chloride (MgCl2) acts as the buffer of the reaction and also as a cofactor to the enzyme taq polymerase. Finally, the deoxyribonucleotides (dNTP's) are the building blocks of new strand of DNA. They are nucleotides in which the taq polymerase synthesizes new nucleootides to base pair with the existing ones of the parent strand. Thermal Cycling Thermal cycling is the process of cycling a PCR tube alternatively in cold or hot temperatures in a matter of different specific times. Thermal cycling is of six steps in which includes the initial step, denature, anneal, extend, the final step, and the final hold. In the initial step, the PCR reaction is first exposed to a high temperature of 95 degrees celsius for 3 minutes. Taq polymerase is activated and begins to read the genetic data from DNA, proofreads, and copies the data or target sequence which it will eventually generate. In the second step denature, due to the fact that DNA is composed mostly of proteins, in high heat of 95 degrees celsius for 30 seconds, proteins denature and start losing their tertiary structure which is the two-stranded structure. The two-stranded structure begins to uncoil and separates into two single-stranded structures of DNA. The next step called anneal is the process in which the contents of the PCR tube is exposed to a temperature of 57 degree celsius for 30 seconds. During this process, and with the single strands, primers begin to attach opposite ends of a strand. The 3' primer attaches to the end of strand that contains the hydroxide group, while the 5' primer attaches to the end with the phosphate group. In the fourth step called extend, the process lasts for 30 seconds and exposed to 72 degrees celsius. The taq polymerase is again activated and searches for primers that are attached to existing strands of nucleotides. Once it finds primers, it will attach new units of nucelotides to the 3' end of the parent strand. Polymerase will not add new nucleotides to the 5' end primer and so the new growing strand will extend from one direction only. Initial Step Denature Anneal Extend Final Step Final Hold 3Nucleotides and Base-pairing
(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the PCR video/ tutorial might be useful. Be sure to credit the sources if you borrow images.)
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