Name: Colby Mark Research and Development Specialist
Name: Hayden McIver Role(s)
Name: Isai Valdez Role(s)
Name: Madison Grayson Role(s)
Name: Andrew Wills Open PCR machine tester
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
(Add image of the full OpenPCR machine here, from the Week 9 exercise. Write a paragraph description for visitors who have no idea what this is)
OpenPCR is a themocycler used to conduct heat through samples of data. This is used to replicate DNA through a series of heating and cooling in order to allow certain chemical reactions to occur.The design is made to prevent any outside reagents from affecting the concentrations of the samples; certain features such as precise temperature control and a heated lid allow there to be no interference with the data. The OpenPCR conveniently connects to your computer and stores the data from every trial.
Experimenting With the Connections
When we unplugged the LCD screen (part 3) from the circuit board (part 6), the machine's LCD screen turned off because power was cut off.
When we unplugged the white wire that connects the circuit board (part 6) to heating block (part 2), the machine showed a drop in temperature.
Test Run
(Write the date you first tested Open PCR and your experience(s) with the machine)
The date that we first tested the Open PCR was on October 23rd, 2013. We didn't think that we would be able to open up the panel and see the inner workings of this machine. We thought it was very interesting to see how the wires are connected and also how the cooling and heating system works with the layered Aluminum plates. The process of completing the entire lab did take quite a long time, but compared to other machines ours worked to perfection.
Protocols
Thermal Cycler Program
DNA Sample Set-up
row 1 cell 1
row 1 cell 2
row 1 cell 3
row 1 cell 4
row 2 cell 1
row 2 cell 2
row 2 cell 3
row 2 cell 4
DNA Sample Set-up Procedure
Step 1
Step 2
Step 3...
PCR Reaction Mix
What is in the PCR reaction mix?
DNA/ primer mix
What is in the DNA/ primer mix?
Research and Development
PCR - The Underlying Technology
Functions of each component of a PCR Reaction
The first step is to gather Template DNA this is a double stranded DNA that is separated and then used to form the replicated DNA. Then primers are added to the solution and these are man-made DNA sequences that contain nucleotides which bind to part of the DNA segment that we want to be replicate. The next step is to add TAQ Polymerase these are sequences of proteins similar to an enzyme that attaches to the primer and adds the nucleotides to the segment where the primers are located in order to replicate the DNA. There are also ways to speed and slow down the replication process, by adding Magnesium Chloride. Magnesium acts as a catalyst to speed up the reaction by binding the taq polymerase to the primers and DNA. There should not be too little as this will cause the binding to slow, or stop all together. If there is too much, the DNA replication process with begin working too fast and could skip sequences, which would lead to errors in replicating the strands. Lastly, Deoxyribonucleotides which are DNA nucleotides base pair with the original DNA strand with the help of the TAQ Polymerase to form copy DNA.
Each step of thermal Cycling
There is the initial step which occurs at 95 degrees Celsius for three minutes. During this step, the double helix DNA structure unwinds. The Denature step which occurs next is at 95 degrees Celsius for 30 seconds. During this step, the template DNA separates into single stranded DNA. After denaturing, the Anneal process starts. This occurs at 57 degrees Celsius for another 30 seconds. During this process, the Taq Polymerase binds and begins to copy the DNA by adding nucleotides. The Final step, which is at 72 degrees Celsius for three minutes, is when the DNA extension or "copying" is completed. The very last process that may or may not be needed is the Final Hold at 4 degrees Celsius. During this step it stops the activity of taq polymerase and prevents unwanted copying.
Nucleotide Base Pairing
DNA is made up of four types of molecules called nucleotides. They are designated as A,T,C, and G. The pairing of these bases is driven by hydrogen bonding, and this allows the DNA strands to stick together. The bases are Adenine (A), which sticks to Thymine (T). And Cytosine (C), which sticks to Guanine (G).
(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
