BME100 f2013:W900 Group16 L4: Difference between revisions
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This experiment began on 10-23-13 at 9:56 am. This ran until 11:39 am. | This experiment began on 10-23-13 at 9:56 am. This ran until 11:39 am. | ||
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The test was completed fairly quickly and the machine seemed to run smoothly. | |||
Revision as of 12:17, 23 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-UPInitial Machine TestingThe OpenPCR machine is a machine that amplifies specific sequences of DNA. The PCR process consists of three main steps which take a total of 1 minute and 30 seconds per cycle. These steps include denaturing, annealing, and extending. During the denaturing step, the machine is at a temperature of 95 degrees Celsius. This temperature causes the DNA to unwind and separate into two separate strands. Then, it the temperature decreases to 57 degrees Celsius in order to go through the annealing process. In this, the primers used to recognize a desired segment of base pairs and tell the DNA polymerase where to start adding base pairs attach to the DNA strands. After this, the temperature slightly increases to the temperature of 72 degrees Celsius. This step is called extending, and the Taq polymerase attaches to the DNA strands in order to build the complementary strand. This process can be ran in a selected number of cycles to produce a specific amount of DNA from a sample. Experimenting With the Connections When we unplugged part 3 from part 6, the machine turns on, but the LED display doesn't turn on. When we unplugged the white wire that connects part 6 to part 2, the white wire doesn't heat up the samples in the heating compartment.
This experiment began on 10-23-13 at 9:56 am. This ran until 11:39 am.
The test was completed fairly quickly and the machine seemed to run smoothly.
ProtocolsThermal Cycler Program
The PCR reaction mix consists of 8 tubes with 50 µL each of Taq DNA polymerase, MgCl2, and dNTP’s.
DNA/ primer mix The DNA/ primer mix consists of 8 tubes with 50 µL each of a different template DNA. All the tubes have the same forward primer and reverse primer.
Research and DevelopmentPCR - The Underlying Technology Function of PCR Components The Polymerase Chain Reaction requires many components to amplify the desired DNA sequence. A DNA template is necessary to begin the entire PCR process because it has a section of interest. The billions of DNA copies that result at the end of PCR are all derived from this original DNA template. Primers are needed to replicate DNA and are used in the normal DNA replication process. The primers used in PCR recognize a desired segment of DNA and bind to the DNA strand after denaturation has finished. By having a specified base sequence segment to bind to, the primers begin the process of targeting and isolating the DNA portion of interest. The job of a primer in DNA replication and PCR is to tell DNA polymerase where to start adding the complementary base pairs to the DNA strand. In PCR, primers are unique because they can be synthetically made to target a specific DNA segment. However, regular human DNA polymerase cannot be used in PCR because it cannot withstand high heats; instead, Taq polymerase, a polymerase taken from a thermophilic bacteria, is used to add the complementary base pairs to the DNA strands. Taq polymerase is used because it can withstand the heat cycles required in PCR. Magnesium chloride is used to help stabilize Taq polymerase which depends on a magnesium concentration to function properly. dNTPs are the single A, T, C, G bases and are the building blocks for the new DNA strands. Steps of PCR The first step of PCR is a 3 minute cycle that heats to 95 degrees Celsius. During this step, the DNA unravels from its double helix formation and lines the two strands of DNA parallel to each other. This step of the PCR aligns DNA properly for the other steps to occur. Denaturation is the next step and lasts for 30 seconds and is still held at the 95 degrees Celsius. In this step, the hydrogen bonds holding the complementary base pairs break and the two DNA strands separate from one another. Annealing lasts for 30 seconds and the temperature drops to 57 degrees Celsius. This is the point in PCR where the primers bind to the DNA strands. After annealing, extension occurs. Extension is a 30 second process held at 72 degrees Celsius in which the Taq polymerase adds the complementary base pairs to the DNA strands starting at the primer. The final step is also at 72 degrees Celsius but lasts for 3 minutes. This step allows Taq polymerase to find all of the primers in the DNA sequences. The last step is the hold. The final hold is at 4 degrees Celsius and is meant for investigating how much DNA is in the sample after PCR has cycled. (Add a write-up, essay-style, organized into paragrpahs with descriptive headers, based on the Q&A's from Section three of your worksheet) (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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