The primary function of the PCR is to change tempuratures rapidly with given set amounts of time.
The PCR machine allows to complete a biochemical reaction called "polymerase chain reaction"(PCR).
The PCR, also known as a thermal cycler, raises and lowers the temperature in the heating block in precise, pre-specified steps in order to amplify the DNA samples in the machine.
Labeled Open PCR
Part 1 - This is the underside of the heating lid. It is also heated, and is pressed against the lids of the sample tubes in order to keep condensation from forming on the inside of the sample lids.
Part 2 - This is the main heating block. This is where the samples are placed before closing the lid and starting the heating and cooling process.
Part 3 - This is the LCD screen. This shows information about the current process when the machine is running.
Part 4 - This is the heat sink. It consists of a number of copper tubes with aluminum fins on them, and a fan to help move the hot air away from the fins. When attached to the heating block, the highly conductive properties of the metals allow the temperature of the heating block to be rapidly reduced.
Part 5 - This is the power supply. It converts alternating current from the wall outlet to direct current for use in the circuitry.
Part 6 - This is the circuit board. It is the part that connects to all the other parts and makes them regulate the heat in the heating block, as well as sending information to the LCD screen. Part 1 Part 2
Part 3, 4, 5, and 6
Images used from ASU BME 100 workbook
Experimenting With the Connections
When we unplugged the LCD screen (part 3) from the circuit board (part 6), the machine would not display anything on the LCD screen.
When we unplugged the white wire that connects the circuit board (part 6) to PCR block (part 2), the machince showed a drop in temperature on the LCD screen.
Test Run
March 20th, 2014, 9:00am Lab
The Open PCR first test run was successful and ran 13 cycles over the span of the lab time. *PASS
After we issued the start command from the computer, the machine sat quietly humming away, with a fan coming on occasionally to help cool the samples. The screen showed the cycle number that it was currently on.
Protocols
Thermal Cycler Program
When beginning a Polymerase Chain Reaction, open the PCR software and add a new experiment.
Change the program specifications as follows:
Heated Lid: 100°C
Initial Step: 95°C for 2 minutes
Number of Cycles: 35
Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds
Final Step: 72°C for 2 minutes
Final Hold: 4°C
DNA Sample Set-up
Positive Control:
Disease DNA
Patient 1, Replicate 1
ID:
Patient 1, Replicate 2
ID:
Patient 1, Replicate 3
ID:
Tube Label:
CP1
Tube Label:
CP2
Tube Label:
CP3
Tube Label:
CP4
Negative Control:
Non-Disease DNA
Patient 2, Replicate 1
ID:
Patient 2, Replicate 2
ID:
Patient 2, Replicate 3
ID:
Tube Label:
XP1
Tube Label:
XP2
Tube Label:
XP3
Tube Label:
XP4
Patient 1: 43849
Patient 2: 80853
DNA Sample Set-up Procedure
Step 1: Label each tube with regards to patient and positive or negative control
Step 2: Use the micro pipette to mix the PCR reaction mix and DNA template with the primer mix
Step 3: Tightly close the lids and place the tubes in the PCR machine
Step 4: Open up the OpenPCR application on your PC and run the machine
PCR Reaction Mix
What is in the PCR reaction mix?
The PCR mix contains Taq DNA polymerase, MgCl2, and dNTP's to amplify the DNA templates.
DNA/ primer mix
What is in the DNA/ primer mix?
There's a different DNA template in each mix. However, every tube has the same forward and reverse primer.
Research and Development
(Add a write-up, essay-style, organized into paragraphs with descriptive headers, based on the Q&A's from Section three of your worksheet)
Function of Components
We can analyze DNA through the Polymerase Chain Reaction. PCR has four separate components: template DNA, primers, taq polymerase, and deoxyribonucleotides(dNTP's). Template DNA's are used as a 'template', or guide, by an enzyme known as polymerase to create a complimentary strand. It is the sample DNA that contains the target sequence and allows the reaction to start. Primers are short pieces of single-stranded DNA that are complementary to the target sequence and serves as the starting point for DNA synthesis. Taq Polymerase is a thermostable DNA polymerase that generates new strands of DNA using the DNA template and primers. Deoxyribonuceotides are the single monomer of DNA (A, T, G, C) that serves as the essential building blocks for new DNA strands.
PCR Steps
Step one : Denaturation - this where the DNA is heated to about 95 degree Celsius to render it single-stranded
Step two : Annealing - the two primers bind the appropriate complementary strand ; the temperature for this step varies depending on the of size of the primer and its homology to the target DNA but it is usually brought down to about 50-65 degree celsius
Step three : Primer Extension - the temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C, and its commonly a temperature of 72 degree celsius is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction
These steps are repeated from 28-35 times. Since the reaction is essentially exponential, and since each cycle is about 5 minutes, a large quantity of DNA can be produced for analysis in as little as few hours.
Base Pairing Adenine pairs with Thymine Thymine pairs with Adenine Cytosine pairs with Guanine Guanine pairs with cytosine
BME 100 Spring 2014
