BME100 s2014:T Group15 L4
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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LAB 1 WRITE-UP
Initial Machine Testing
An OpenPCR machine is an open-source, hackable, DIY PCR machine. PCR stands for Polymerase Chain Reaction, and it's an important tool for just about any type of modern molecular biology. It works by amplifying a specific part of a strain of DNA in order to study that section more carefully.
Experimenting With the Connections
When we unplugged (part 3) from (part 6), the machine's LCD screen turned off.
When we unplugged the white wire that connects (part 6) to (part 2), the machine the temperature displayed on the LCD screen dropped from 25 degrees C to -40 degrees C.
(Write the date you first tested Open PCR and your experience(s) with the machine)
We opened the OpenPCR program, added a new experiment, and entered the following parameters:
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
EXTEND at 72°C for 30 seconds
FINAL STEP: 72°C for 2 minutes
FINAL HOLD: 4°C
We then placed the empty tubes in the OpenPCR machine, turned the knob on the top until there was some resistance which indicated the top was touching the tubes. Next we turned the machine on and pressed the start button on the OpenPCR program. The LCD screen displayed which run it was on and which step within that run. Our OpenPCR machine was a pass, meaning it ran with no problems.
Thermal Cycler Program
The mix contains taq DNA polymarase, MgCl2, and dNtp's.
DNA/ primer mix
Each mix contains a different template DNA. All tubes have the same foward primer and reverse primer.
Research and Development
PCR - The Underlying Technology
(Add a write-up, essay-style, organized into paragraphs with descriptive headers, based on the Q&A's from Section three of your worksheet)
DNA is analyzed with PCR reactions with the help of four main components: Template DNA, Primers, Taq Polymerase, and Deoxyribonucleotides (dNTP's). The template DNA is the original strand of DNA, and contains the section that is to be replicated via PCR. The primers attach to the template strand in two places in order to indicate to the DNA Polymerase where to begin and end replication. Taq polymerase handles the replication of the indicated strand by adding on a complimentary set of bases, which are the dNTP's, in between the primers.
In order to replicate the strand of DNA via PCR, first the template strand is heated to 95 degrees Celsius for 3 minutes in order to begin denaturing. The denaturing continues at 95 degrees for 30 seconds, at which point the template DNA strands are fully separated. The annealing process then begins at 57 degrees Celsius for 30 seconds, which allows the primers to attach to the complementary strands of DNA. This process is extended at 72 degrees Celsius for 30 seconds, which is when the Taq polymerase binds to the primers. The final step is held at 72 degrees for 3 minutes, and the Taq polymerase attaches the nucleotides to create a complementary strand of the copied template DNA.
(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.)