Name: Ojeen Korkes Research and Development Scientist
Name: Brandon Simmons Open PCR Machine Engineer
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
This is an image of an Open PCR Machine. This machine regulates the temperature of the DNA smaples both heating and cooling samples to the preset temperature and times set up beforehand using the set up program. This heating and cooling seperates the DNA strands at high heat and then allows it to synthesize at low temperature.
Experimenting With the Connections
When we unplugged part 3, the LCD, from part 6, the Open PCR circuit Board, the LCD on the machine turned off and no information appeared on the LCD screen.
When we unplugged the white wire that connects part 6, the Open PCR circuit Board, to part 2, the heat sink, the machine no longer accurately regulates and controls the temperature and result in malfunction of heating the PCR tubes.
Test Run
On October 25 2012, we conducted our first test on our open PCR machine. We tested the machine to test the operation functionality. The initial test demonstrated the machine heat sink accuratly controlled and displayed the preprogrammed temperaute determined by the software on the computer. The overall successfullness of the machine was good, however it came with one difficulity, fluctuation of time to complete the preporgrammed cycles.
Protocols
Polymerase Chain Reaction
PCR, polymerase chain reaction, is a simple tool that one can use to focus in onto a segment of DNA and generate thousands to millions of copies of a particular DNA sequence. In PCR, everything relies heavily on the regulation and variation of temperature. By setting the PCR at specific temperatures, a chain of reactions can take place. Therefore, there are three steps in PCR cycle: denaturation, annealing, and extension. In the beginning, PCR should be heated to 95 degrees Celsius. This allows the double-stranded DNA to be separated and unwinded. Secondly, PCR should be cooled at 57 degrees Celsius because this allows a piece of DNA to bind to DNA product from the initial step. This is done through primer enzymes that allow polymerase to start synthesizing by recognizing and attaching to sequences that are complementary. Lastly, the extension step is where DNA product will continually add bases following the primers until it fully synthesizes a new strand of DNA. This last step is done at 72 degrees Celsius. In the end, the output of these reactions yield up to millions of strands of DNA that can be examined to identify certain types of genes in diseases or utilized for scientific purposes.
Steps to Run PCR
Connect the PCR machine to the computer.
Open the 'OpenPCR' program on the computer.
Label the tubes. This information should include the patient number (1 or 2) or control (+ or -), as well as the replication number (1, 2, or 3).
Prepare the experiment by inserting the reactants into the PCR tubes. These tubes will consist of the patients DNA, along with the other provided mixing components*. After filling each tube, put it into the chamber at the top of the machine.
Close and tighten the lid of the chamber.
Customize the settings in the 'Thermal Cycler' program to include three stages: Stage 1 - one cycle that will heat the reactants up to 95 degrees Celcius for three minutes, Stage 2 - 35 cycles that will heat the reactancts to 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, and 72 degrees Celsius for 30 seconds.
Press start on the prgram to begin running the PCR.
Collect and record data at the completion of the trial.
* Provided Mixing Components
a) TaqDNA polymerase (non-recombinant modified form)
b) MgCl2
c) dNTP's
d) reaction buffers (at optimal concentration for DNA template amplification)
Reagent
Volume
Template DNA (20 ng)
0.2 μL
10 μM Forward Primer
1.0μL
10 μM Reverse Primer
1.0μL
GoTaq master mix
50μL
dH2O
47.8 μL
Total Volume
100.0 μL
The Patient Information
Patient Identification Number
Gender
Age
31542
Male
55
52125
Female
55
Flourimeter Measurements
Flourimeter Assembly Procedure
Place a glass slide on the device.
Using a pipette, add two drops of water to the slide.
Turn on the blue LED light, and adjust the slide so that the light shines directly through the center of the water drop.
Adjust the camera settings on a smartphone as follows:
Turn off the flash
Set exposure to the highest setting
Set saturation to the highest setting
Set contrast to the lowest setting
Place the smartphone on the phone holder and position it in front the of fluorimeter device.
Cover the entire setup with a black box in order to create as dark of an environment as possible.
Take a picture with the smartphone. For best results, set the camera timer on the smartphone in order to be able to take a picture with the box completely closed.
How to Open Pictures Using Image J
Using the smartphone, email the images to someone in the group with a computer.
From the computer, open the email and download the images.
In Image J, go to file, open, and then select the desired picture.
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
In Week 3 of our PCR experiment, we studied the cancer marker in the PCR experiment was correlated to the breast and colorectal cancer. Based on conditional probabilities, we found that the frequency of this cancer found in Finland was 7.8%.
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 OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.
Summary: This is an image of how RNA primers bind to the cancer DNA template during the replication process. [1]
BME 103 Fall 2012
![[1]](http://matthew2262.files.wordpress.com/2011/04/replication.jpg){kind=link}
![[2]](http://www2.le.ac.uk/departments/emfpu/genetics/explained/images/PCR-process.gif){kind=link}