Name: Amber Bengson Role: Open PCR Machine Testing
Name: Chandler Varone Role: Open PCR Machine Testing
Name: Prycilla Jones Role: Protocol Planning
Name: Emily Mei Role: Protocol Planning
Name: Terri Bivins Role: Research and Development
LAB 1 WRITE-UP
The Original Design
The Pictures above are of an OpenPCR machine. What this is, is a user assembled PCR machine, the machine performs a certain
number of cycles that the user adjusts. The temperature that is desired by the user can be set for each cycle. You connect the
OpenPCR machine to a computer and use the software to complete the cycles.
Experimenting With the Connections
Part 3 )When we unplugged the wires, coming from the screen, from the "brains/motherboard", the machine's screen went blank.
Part 6) When we unplugged the white wire, that comes from the thermometer, from the "brains/motherboard", the machine read
-40 degrees Celsius. When the wire was plugged in, it also read -40 degrees Celsius
"Test Run"
When we started the PCR machine it was 12:53PM on Wednesday the 23rd. It was on the first cycle until 1:31PM at that time
the instructor told us to unplug the machine because it was not working. This was the first time we had worked with this
type of PCR machine, and it was a bad experience. We would have hoped that the machine would have been able to
complete its cycles.
Procedure:
Step 1: Determine positive and negative control. The negative control will not have the cancer marker. The positive control has cancer marker.
Step 2: Label Tubes.
Positive control: cancer Negative control: Not cancer
Patient 1 43849 Patient 2 80853
1A 2A
1B 2B
1C 2C
Step 3: Add DNA/primer mix to positive control.
Step 4: Add PCR reaction mix to positive control.
Step 5: Place sample in Open PCR(thermo cycler)
Step 6: While in the Open PCR the sample will be heated up to 95℃ the DNA chain separates into two strands. The temperature then decreases to 50℃ and the primers attach to the separate strands at the target sequence. The polymerase attaches to the primer and begins to replicate the strand.
Step 7: Repeat steps 3-5 for the remaining tubes.
Step 8: Compare Patient 1 DNA replicates to those of the positive and negative controls.
step 9: Compare Patient 2 DNA replicates to those of the positive and negative controls.
PCR Reaction Mix
-Taq DNA Polmerase
-MgCl2
-dNTP's
DNA/ primer mix
What is in the DNA/ primer mix?
Research and Development
PCR - The Underlying Technology
1PCR 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 an enzyme copies from. The Taq Polymerase is a thermostable enzyme. It reads the genetic information form the DNA template and checks for mutations before copying data.
2Thermal Cycling
Thermal cycling is the process of cycling a PCR tube holding the PCR reaction in specific variations of time and temperature. The PCR reaction is first exposed to high temperatures, it is lowered, and increases slightly in temperature. enzymes and/or reagents and is done ...
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.)
BME 100 Fall 2013
