BME100 f2013:W1200 Group3 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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Contents

OUR TEAM

Name: Samuel MokdadRole(s) Team Leader/ Research & Development
Name: Samuel Mokdad
Role(s) Team Leader/ Research & Development
Name: Morgan JamesonRole(s) Initial Machine Testing
Name: Morgan Jameson
Role(s) Initial Machine Testing
Name: Tony FacchiniRole(s) Research & Development
Name: Tony Facchini
Role(s) Research & Development
Name: Cameron GhodsRole(s) Initial Machine Testing
Name: Cameron Ghods
Role(s) Initial Machine Testing
Name: Evan EppersonRole(s) Protocols
Name: Evan Epperson
Role(s) Protocols

LAB 4 WRITE-UP

Initial Machine Testing

The Original Design

Image:PCRmachine1.jpg

The OpenPCR is a technique used to copy DNA molecules. This processs is done by dramatic tempertaure changes to isolate DNA strands from one another. To begin the machine is heated to almost boiling to denature the two strands from the original DNA. Once the two strands are seperated, the temperature decreases to allow the primers to attach and bind to complimenterary matches of DNA. This initiates the coping process and the taq polmerase then attahces (slightly higher tempertaure) to add nucleotides to the sequence to extend the strand. The process is completed with the four strands of DNA. This is the first cycle and many more occur to have billions of strands duplicated. The Heating plate is responsible for heating the PCR samples. The function of the LCD display is to show the current progress of the machine and ensure the users that the machine is working. The cooling fan is self explanatory, it simply cools the samples being tested. Circuit boards are the brains of the machine; they make sure everything is powered and works at the correct time.

Experimenting With the Connections

When we unplugged part 3 from part 6, the screen on the machine turned off.


When we unplugged the white wire that connects part 6 to part 2, the machine would not power on and it stopped.


After letting the machine run for about an hour, it was still on the first cycle. This was done on October 23,2013.




Protocols

Thermal Cycler Program


DNA Sample Set-up

Patient 1 ID: 77842 Tube: 1A Patient 1 ID: 77842 Tube: 1B Patient 1 ID: 77842 Tube: 1C Patient 1 ID: 77842 Tube:+ Control
Patient 2 ID: 26204 Tube: 2A Patient 2 ID: 26204 Tube: 2B Patient 2 ID: 26204 Tube: 2C Patient 2 ID: 26204 Tube: - Control


DNA Sample Set-up Procedure
Step 1: Prepare the DNA/Primer mix and the PCR Reaction mix and well as the controls (both positive and negative)
Step 2: Label the test tubes

Step 3: Add 50μL of the DNA sample Mix to each of the correspondingly labeled reaction test tubes. *Be sure to use new pipettes each time*

Step 4: Start the PCR machine and wait approximately 105 minutes while the machine goes through 35 cycles based on the set-up shown directly below.
HEATED LID: 100°C
INITIAL STEP: 95°C for 3 minutes
STEP: 35 cycles, 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 3 minutes
FINAL HOLD: 4°C
Step 5: Analyze the results


PCR Reaction Mix

  1. Taq DNA Polymerase
  2. dNTPs
  3. MgCl2


DNA/ primer mix

  1. 3μL Forward Primer
  2. 3μL Reverse Primer
  3. 4μL Fluorescent Probe (specific)
  4. 5μL Sample of each patient's DNA





Research and Development

PCR - The Underlying Technology

Components of Polymerase Chain Reaction:

There are many components which all play a part in the process of Polymerase Chain Reactions. First, the original DNA strand is used as a template from which millions of copies are made during the process of PCR. Once the DNA sequence has been separated, the DNA primers will bind to both strands. These primers will bind to specific starting sequences or starting codons. Once both primers bind to the two template DNA strands, the Taq Polymerase bind to the end of the primer sequence. The Polymerases then add complementary nucleotides to the growing replicant strand until they reach a stop codon. Magnesium Chloride is needed in this step because Taq Polymerase is a Magnesium (Mg 2+)dependent enzyme. The Taq Polymerase is able to add nucleotides to the growing strand because dNTP's, a solution containing free-floating base pairs, is present in the solution.

Polymerase Chain Reaction Procedure:

The process of Polymerase Chain Reactions contains six main steps, three of which are repeated about 35 times. The first step is to prepare the PCR reaction device by keeping the heating block at 95 degrees Celsius for three minutes. After this step, the cyclic portion of the experiment begins. The template strand is heated to 95 degrees Celsius for 30 seconds. In the absence of DNA helicase to split the strands, instead this method supplies the strand with enough kinetic energy to split the double strand into the two complementary strands. The temperature of the reaction vessels is then lowered to 57 degrees Celsius for 30 seconds. This step, called annealing, is the optimal temperature in which the primers will attach to the split DNA strands. The annealing step sets up the extending step. In this step, the temperature is increased to 72 degrees Celsius. At this specific temperature, the Taq Polymerase enzymes add complementary nucleotides to the ends of the primer sequences. The denaturing, annealing, and extending procedures are repeated as necessary. On the last extending cycle, the temperature is left at 72 degrees Celsius for another three minutes. This ensures that the Taq Polymerase enzymes have enough time to finish adding complementary base pairs to all of the newly created DNA segments. Finally, the solution is held at 4 degrees Celsius in order to keep the solution at an appropriate temperature for short-term storage until other tests can be run on the newly amplified DNA.

Extra Credit:

Image:PCR_Process.png








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