BME100 f2013:W900 Group5 L4

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BME 100 Fall 2013 Home
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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: Lincoln(Grady) BainRole: Open PCR Machine Testing
Name: Lincoln(Grady) Bain
Role: Open PCR Machine Testing
Name: Andrew Olson
Name: Andrew Olson
Name: Pedro Giorge
Name: Pedro Giorge
Name: Niko Vlastos
Name: Niko Vlastos
Name: Omar Alsubhi
Name: Omar Alsubhi

LAB 1 WRITE-UP

The Original Design
Image:OpenPCR_solo-W490.jpg
This image shows an Open PCR machine, which is an affordable thermocycler that can perform polymerase chain reactions that amplify certain segments of dna, by using variance in temperatures to trigger certain mechanics. The process essentially replicates a single strand of DNA into multiple copies, however it must be used with a USB port and a computer through the downloadable program OpenPCR. This is how the temperature and the number of cycles can be adjusted accordingly, and all the information is downloaded straight into the computer itself, which makes data and result collection much more efficient.

Experimenting With the Connections
Image:PCRinsides.png

After unplugging part 3 from part 6, we had discovered that the ,machines LCD screen was part 3 and the circuit board is part 6, and when that line was severed, the LCD screen turned off completely. Then when we decided to unplug part 2 and part 6 from each other heating was completely changed from one constant to another in degrees Celsius and essentially turned the heating component off within the open PCR machine. Part 2 is the heating device/core and part 6 is the circuit board and when they were unplugged the device when from 26.7 degrees Celsius to -40 degrees Celsius. Obviously all the connections to the circuit board hold a drastic importance to the Open PCR device.


Test Run The first time using an Open PCR machine was rather lack luster do to the fact that the group allowed for the machine to run for nearly a two hour period and the device barely made its way to the 6th cycle. The device was from that point deemed slow and returned to the front desk, only to be used again shortly.

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Protocols

The purpose of protocol planning Protocol planning is important for many reasons, a few of which are elementary. Critical planning is put in place as the backbone that one will build upon. In other words, it is an outline of what should be done within a study or experiment or even simple things like how to label test strips. Without protocol planning experiments would contain too many variables and it would be much easier to make a mistake. Performing a study without protocol planning is like playing soccer without the rules. i.e. Protocol planning in this analogy would be the rules. In this lab we discussed the protocol for labeling test tubes along with the protocol of how to use the open PCR equipment. Protocol planning is of the utmost importance especially in a lab or while doing a study.

Thermal Cycler Program


DNA Sample Set-up

positive control cancer DNA template patient 1 ID 32298 patient 1 ID 32298 patient 1 ID 32298
table label CNC table label 101 table label 102 table label 103
negative control non-cancer DNA template patient 2 ID 26685 patient 2 ID 26685 patient 2 ID 26685
table label CNC table label 201 table please 202 table label 203


DNA Sample Set-up Procedure

  1. 1 plug in the CPR Machine.
  2. 2 pipette the eight samples into the corresponding test tubes.
  3. 3 bad the primer mix to each using a pipette.
  4. 4 at the reaction mixture the same way
  5. 5 please test into the openPCR Machine
  6. 6 start the machine
  7. 7 after the open PCR machine has run its course. Store test tubes for future use.


PCR Reaction Mix

  • What is in the PCR reaction mix?

50 ul each Tap DNE polymers-the protein responsible for assembling the newly created strands. MgCl2 dntp's

DNA/ primer mix

  • What is in the DNA/ primer mix?

50 ul each DNA template (different for each trial) Forward primers (same for each trial) Reverse primers (same for each trial)





Research and Development

PCR - The Underlying Technology

What are the functional components of PCR?

Template DNA is the a portion of the DNA that contains the target vector. There are two different primers that are attacked to are made to match each segment of DNA and copy it. One primer will attack to the top strand of the DNA and the other will attack to the bottom strand of DNA. The taq polymerase is an enzyme the synthesizes DNA to be complimentary to a target strand. New Strand can be generated by using the templates and primers. Magnesium chloride is used as a catalyst and the more MgCl that is used, the faster the reaction will occur. The different types of nucleotides are A,T,C,G.

What are the different nucleotides?

The different nucleotides are adenine, thymine, guanine and cytosine and the base pairs match up and arrange them selves in the fashion A<->T C<->G

What are the phases of cycling?

The primary step is for the thermo cycler to start at 95 degrees Celsius for 3 min, which separates the single strand template. Next step also retains the same amount of heat but for only 30 seconds to allow for the machine to denature the DNA. Next, 57 degrees Celsius for 30 seconds followed by 72 degrees Celsius to activate the DNA polymerase, which locates a primer and attaches itself. The final step for the cycle is to sit at 72 degrees Celsius to allow complementary binding, followed shortly by 4 degree Celsius to end the thermal cycling.


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