BME100 f2013:W1200 Group1 L4
|Line 78:||Line 78:|
4°C: ''The PCR reaction ends.''
4°C: ''The PCR reaction ends.''
Revision as of 03:16, 29 October 2013
|BME 100 Fall 2013|| Home |
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
Wiki Editing Help
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
The image shown above is of a device called an OpenPCR machine. An OpenPCR machine is a low cost, yet highly accurate thermocycler capable of performing polymerase chain reactions that amplifies a specific target DNA segment while maintaining extreme differences in temperature change. After going through many cycles of heating and cooling, the OpenPCR machine replicates a single strand of DNA into many copies. As seen in the image, the OpenPCR machine is connected to a computer using a USB cable. The computer uses an OpenPCR program that allows the number of cycles and each step and desired temperature and time to be adjusted accordingly. Thus, the OpenPCR machine begins the process according to the information inserted into the program.
The image shown above is of the inside of the OpenPCR machine. The environmental changes of heating and cooling are controlled by the heater, heating lid, and cooling fan. The data collected is computed and transferred from the circuit board to the LED screen on top of the OpenPCR machine.
Experimenting With the Connections
The PCR machine is heavily dependent on numerous connections and and interconnected parts. This means that, in order for it to work well, everything must be in its proper location and plugged in. This being said, when we unplugged part 3 (the LCD screen) from part 6 (circuit board) of the machine, the connection from the LCD to the circuit board was severed, resulting in the screen turning off. Also, when we unplugged the white wire that connects part 6 (circuit board) to part 2 (core) of the machine, the temperature changed from 26.7 degrees Celsius to -40 degrees Celsius. It is very important that all connections are correct in order to have an effective PCR machine.
Thermal Cycler Program
95°C for 3 minutes: The initial Template DNA strand separates.
95°C for 30 seconds: The DNA double helix separates, creating two single-stranded DNA molecules.
57°C for 30 seconds: The two primers attach to opposite ends of the top and bottom strand of the target DNA segment.
72°C for 30 seconds: The DNA polymerase activates. Replication of the target DNA segment begins.
72°C for 3 minutes: The complementary binding of nucleotides continues until it gets o the end of the DNA strand and falls off.
4°C: The PCR reaction ends.
The PCR reaction mix will contain 8 tubes. Each tube will contain 50μL of the following:
The DNA primer mix contains 50μL of the following:
Research and Development
PCR - The Underlying Technology
Template DNA is the sample DNA that contains the target DNA segment. Two primers are designed to match to the segment of desired DNA to copy. One primer attaches to the top strand at one end of the target DNA segment and the other primer attaches to the bottom strand at the opposite end. Taq Polymerase is an enzyme that synthesizes new strands of DNA complimentary to the target strand. It can generate new strands of DNA using a DNA template and primers. It is heat resistant. Magnesium Chloride is how magnesium is added to the PCR reaction. It acts like a catalyst. It is not consumed in the reaction but is a necessary component. The more Magnesium Chloride is added, the faster the reaction goes. Deoxyribonucleotides provide the energy and nucleotides for the synthesis of DNA. The nucleotides include Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). They are collected by the Taq Polymerase and used in complementary sequencing of the template DNA strand.
What happens during each step of thermal cycling?
The initial step of thermal cycling takes place at 95°C for 3 minutes. In this step, the single-stranded template DNA separates. The next step also takes place at 95°C but for 30 seconds and involves the denature of the double-stranded DNA. Each double-stranded DNA separates due to the high heat into single-stranded DNA segments. Next, anneal occurs at 57°C for 30 seconds. The thermal cycler cools down. When this happens, the single-stranded DNA segments try to pair up. One of the primers attach to the top strand of DNA at one end while the other primer attaches to the bottom strand on the opposite end of the DNA segment. As the thermal cycler heats back up to 72°C, the step of extending occurs for 30 seconds. The added heat activates DNA polymerase. Taq Polymerase locates a primer attached to the single-stranded DNA segment and adds complementary nucleotides. The final step of thermal cycling occurs at 72°C for 3 minutes. The complementary binding of nucleotides continues until it gets to the end of the strand and falls off. The final hold of the process happens at 4°C. This step ends thermal cycling.
Which base sticks to each base listed below?
Adenine (A) is paired up with the base Thymine (T). Thymine (T) is paired up with the base Adenine (A). Cytosine (C) is paired up with the base Guanine (G). Guanine is paired up with the base Cytosine (C).