BME100 f2013:W900 Group4 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: Philip TerzicResearch and Development
Name: Philip Terzic
Research and Development
Name: Elliott TejadaOpen PCR Testing
Name: Elliott Tejada
Open PCR Testing
Name: Sierra Headrick Protocol Planner
Name: Sierra Headrick
Protocol Planner
Name: Aaron BlankOpen PCR Testing
Name: Aaron Blank
Open PCR Testing
Name: Alexandra TaylorOpen PCR Testing
Name: Alexandra Taylor
Open PCR Testing

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:OPEN_PCR.jpg Image from: practicalhorsegenetics.blogspot.com
The open PCR machine shown above is used to copy and amplify small samples of DNA. In order to copy the DNA, the machine first heats up to 95 degrees Celsius to separate the DNA strands. Following the machine cools to about 55 degrees Celsius so that DNA primers can latch to the DNA strands. Next, the machine will heat up to about 72 degrees Celsius so that an enzyme can attach to the DNA primers and create the other halves of the initial strands of DNA. These steps are all part of one cycle and the open PCR machine can run more cycles depending on what the desired amount of DNA is. Also, this machine uses a computer app that allows the user to edit the specific amount of cycles that occur and the exact temperatures that the machine will heat up and cool to.


Experimenting With the Connections

When we unplugged LED display from the Brain Board, the machine began flashing and fluctuating several different patterns, numbers, and letters on the LED display. When we unplugged the white wire that connects Brain Board to the LED display, the machine loses the ability to take the correct temperatures and went from the starting temperature in Celsius down about sixty degrees.

Test Run

On October 23rd, 2013, at approximately 10:17 AM, the OpenPCR machine was started and ended at about 11:35 due to the time limits. The time remaining in the machine's cycle was about 35 minutes. When working with this machine we did experience some flaws in the construct such as loose panels and hard to reach places. Also the small screws did not have the nut to go along with most of them. This left the walls of the machine unstable and not secure when examining the machine. Other than those minor flaws, our experience with the OpenPCR machine was quite satisfying.




Protocols

Thermal Cycler Program
The thermal cycler program used in this laboratory will be the OpenPCR Open Source Thermal Cycler. After performing a calibration procedure in the previous lab section, the Thermal Cycler Program is set up to perform the steps of Polymerase Chain Reaction (PCR) in roughly 2 hours according to the manufacturer's manual. The thermal cycler will raise its heat to ~95 degrees and the sample will undergo denaturing where the two strands of DNA separate from one another (denaturation). After separation, the temperature will lower to ~57 degrees where the primers will bind to their complimentary matches on the target DNA (annealing). Finally the temperature will raise once more up to ~72 degrees and the taq polymerase will add nucleotide bases to the second strand (extension).

DNA Sample Set-up

Positive Control: Cancer DNA Template (Label = P) Patient 1, Replicate 1 ID:89941 (Label = A) Patient 1, Replicate 2 ID:89941 (Label = B) Patient 1, Replicate 3 ID:89941 (Label = C)
Negative Contro: Non-Cancer DNA Template (Label = N) Patient 2, Replicate 1 ID:59749 (Label = 1) Patient 2, Replicate 2 ID:59749 (Label = 2) Patient 2, Replicate 3 ID:59749 (Label = 3)

DNA Sample Set-up Procedure

  1. The first step for setting up the DNA samples will be to go through and label each of the thermal cycling tubes adequately and so there is no confusion throughout the course of the lab.
  2. With tubes adequately labelled, 50μL of the "PCR Reaction Mix" will be pipetted into each of the eight PCR reaction tubes.
  3. As an added precaution, the disposable pipette tips will be removed and thrown away between each sample so that there is no chance of cross contamination that would add error to the data.
  4. In the same fashion as the second and third steps, 50μL aliquots of the "DNA/Primer Mix" will be added to each of the eight reaction tubes and the pipette tips will again be disposed of between each sample.
  5. At this point, the samples are all placed in the correct tubes. They should be mixed thoroughly, some PCR experiments will run their tubes through a centrifuge to ensure complete mixing.
  6. Mixed samples are ready to be placed into the thermal cycler.


PCR Reaction Mix

  • The PCR Reaction Mix contains Taq DNA Polymerase, MgCl2, and dNTP's.


DNA/primer mix

  • The DNA/primer mix contains the template DNA with both a forward and a reverse primer that will attach to the target region on the DNA.

Below is a screen shot of the correct OpenPCR program set to the correct parameters and ready to run:




Research and Development

PCR - The Underlying Technology

There are five basic components of a Polymerase Chain Reaction, each with specific functions that help complete the process. The template DNA is the original strand of DNA that will then be replicated to create new strands. Primers are sequences of DNA that attach to the start and end of the specific strand trying to be replicated. Taq Polymerase reads the DNA sequence and attaches matching nucleotides to the original strand to build a copy. During the PCR reaction, Magnesium Chloride causes the reaction to go forward and determines the speed of the reaction. It is a cofactor of DNA Polymerase and must be optimized for each primer. Deoxyribonucleotides, on the other hand, are single nitrgen-base units that act as the building blocks of DNA.

After the PCR reaction mix, the sample and the DNA/Primer mix have been combined, and the sample has been placed into the 16 tube PCR block, the thermal cycler will go through an initial step where it heats up to ~95 Degrees Celsius and is then held at this temperature for 3 minutes to begin the process of activating the DNA polymerase. After this step is complete, the denature step begins --this also occurs at ~95 Degrees Celsius-- and during this step, the sample DNA will separate into single strands. From here, the temperature will be decreased to ~57 Degrees Celsius for the annealing phase. During this phase, the two primers will bind to their complimentary base pair on the target DNA. The temperature will be raised once more but to ~72 degrees Celsius for the extension phase. During the extension phase, the taq polymerase will bind to the primer and will begin adding the four nucleotides described below to the second strand of DNA. According to the given hand out, the denaturation, annealing and extension phases each occur for 30 seconds. the ~72 degrees will be held for 3 more minutes after the extension step in order to ensure that all of the single strands have been fully extended. Finally the temperature will be lowered to ~4 degrees Celsius and the data collected from the thermal cycler will be stored for reference.

DNA is built using four types of nucleotides, A, C, T, and G. These bases stick to corresponding bases on the second strand of the DNA via hydrogen bonding, and allow for the attachment of primers to stick to the template strand. Adenine sticks to Thymine, Thymine to Adenine, Cytosine to Guanine, and conversely Guanine to Cytosine.

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