BME100 s2014:T Group14 L4

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BME 100 Spring 2014 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: Caitlin R. Byrne
Name: Caitlin R. Byrne
Name: Megan McGuire
Name: Megan McGuire
Name: Wade Savage
Name: Wade Savage
Name: Theo Hall
Name: Theo Hall

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

OpenPCR Machine

A PCR machine is basically a photocopier for DNA. It splits apart the double helix by heating in order to make copies of it. For a polymerase chain reaction to take place a particular DNA sequence should be identified and then amplified by creating up to millions of copies of the specific strands. A PCR machine can go through many cycles of this process and can turn a strand of DNA into thousands of copies of the exact same single strands of DNA. The OpenPCR uses a display on the actual device and a USB port to connect to the computer in order to change the parameters of the PCR reaction we want to take place. The OpenPCR machine is basically a "Do-It-Yourself" kit for anyone to buy, make, and then use. Since it's you have to put together, this equipment is open to some errors if not put together right. This is why we tested the PCR machines before conducting our experiment.

Experimenting With the Connections

When we unplugged (part 3) from (part 6), the machine's display turned off; this is the power cable for the display.

When we unplugged the white wire that connects (part 6) to (part 2), the machine's display showed an incorrect temperature. We assume that this is the wire that connects the display and internal computer to the integral part of the device that measures the temperature of the PCR machine.


Test Run

We preformed our test run of our OpenPCR machine on Thursday March 20, 2014. Set up was simple and went smoothly, we placed the empty test tubes into the machine and ran the machine. After setting up everything correctly, our machine ended up failing the test. The computer program projected that our the time for our reaction would extend way beyond the two hour time period and didn't even finish 1 cycle. This resulted in our machine's failure and was noted to not be used during the experiment.




Protocols

Original image of our group's inputs into the OpenPCR software.
Original image of our group's inputs into the OpenPCR software.

Thermal Cycler Program

  1. Make sure the OpenPCR machine is connected to the computer through the USB cord.
  2. Start Open PCR Software
  3. Select "Add a New Experiment"
  4. Click "More Options" to begin inputing the specifications of the cycles the PCR machine will go through.
  5. Enter Program Parameters:
    • HEATED LID: 100°C
    • INITIAL STEP: 95°C for 2 minutes
    • NUMBER OF CYCLES: 35
    • 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 2 minutes
    • FINAL HOLD: 4°C

DNA Sample Set-up

This is the test tube set up we inserted into the OpenPCR system. We had two different subjects of DNA, one female and one male, and we labeled everything as follows on our 8 50 microliter test tubes.

Patient 1 Patient 2 Controls
Sample 1 1a 2a dDNA (Positive Control)
Sample 2 1b 2b ndDNA
Sample 3 1c 2c

dDNA -- the disease DNA

ndDNA -- the non-disease DNA

female subject -- 1a, 1b, 1c

male subject -- 2a, 2b, 2c


DNA Sample Set-up Procedure

1. Collect materials needed, which include: the PCR reaction mix in 8 test tubes with 50μL in each, 8 empty attached PCR tubes, 8 tubes of the Template DNA and the primer mix combined with 50μL in each tube, a box of disposable pipette tips, and a 200μL micropipettor. 2. Then cut the tubes in sets of 4. You need to do this in order for the tubes to fit in the OpenPCR machine. 3. Then the tubes will be labeled with the predetermined labels we named above with a permanent marker. 4. The tubes are placed in a test tube rack after being labeled. 5. Attach a tip to the micropipettor, and transfer 50μL of the PCR reaction mix into the positive control. Then, dispose the tip into the proper waste disposal. 6. Transfer the positive disease containing DNA and primer mix into the same tube and use a new tip and dispose it properly. 7. Repeat steps 5 and 6 for the negative control, patient 1, and patient 2, remembering to dispose the tip after each substance in each PCR tube. In total 100μL will be each test tube. 8. Place the tubes into the PCR machine, set up the parameters needed for the test run. 9. Get help from the TA in order to start the machine.

PCR Reaction Mix

  • What is in the PCR reaction mix?
  • Taq DNA polymerase, MgCl2, and dNTP's.

DNA/ primer mix

  • What is in the DNA/ primer mix?
  • template DNA, and the same forward primer and reverse primer.




Research and Development

PCR - The Underlying Technology

(Source credit goes to group 3 PCR)

Components:


LCD Screen:This is for the user to observe the status of the PCR machine. Temp, cycle and status can be read on the screen.

Heat Sync and Fan: The Heat Sync is the source of heat for the PCR testing. This piece is directly connected to the loading tray for the DNA samples. The fan is there to keep the insides of the PCR machine from overheating and failing.

Power Supply: The power supply directs power to all components of the PCR machine.

Motherboard/Brain Board: This piece holds all of the working of the PCR machine. The programming and functions of the PCR machine are carried out by the circuitry and the display of the LCD screen relies on this.

Polymerase Chain Reaction:

DNA Template: Strand of DNA that holds the target sequence for PCR testing.

DNA Polymerase: An enzyme that synthesizes strands of DNA. Taq DNA polymerase is commonly used. This enzyme is used for its heat resistance and can withstand the energy required for PCR targeting.

Primers: Primers are single strands of protein that attach the the split target DNA sequence and make way for the copying of the DNA sequence.

Nucleotides: Basic blocks of DNA (A,T,G,C) used for copying the target sequence of DNA.

This is a basic visual representation of a polymerase chain reaction that we made ourselves on PowerPoint.  After identifying what section of DNA you wish to copy, the PCR machine splits apart the double helix structure in order to add additional base pairs that match the old half of the DNA.  This can result in as many copies of DNA as you wish, doubled each cycle.
This is a basic visual representation of a polymerase chain reaction that we made ourselves on PowerPoint. After identifying what section of DNA you wish to copy, the PCR machine splits apart the double helix structure in order to add additional base pairs that match the old half of the DNA. This can result in as many copies of DNA as you wish, doubled each cycle.


PCR Steps The PCR alternates heating and cooling the PCR sample through three temperature steps: denaturing, annealing, and synthesis. The first step, denaturing, involves high heat around 95 degrees Celsius used to separate the double-stranded DNA. In the second step, the temperature is lowered so that the oligonucleotide primers can anneal to the single-stranded DNA. One primer anneals to the left side of one of the DNA strands, while the other primer anneals to the right side of the complementary strand of DNA. In the third and final step, the temperature is raised once again, this time to a temperature around 75 degrees Celsius, so that the polymerase can extend each primer and synthesize the DNA.


DNA Base Pairing Explained

In this image you can see how the base pairs connect through hydrogren bonds.  As always, Adenine corresponds to Thymine and Guanine corresponds to Cytosine.  You do not see the backbone of DNA in this picture though.  Source: http://www.bio.miami.edu/~cmallery/150/gene/c16x6base-pairs
In this image you can see how the base pairs connect through hydrogren bonds. As always, Adenine corresponds to Thymine and Guanine corresponds to Cytosine. You do not see the backbone of DNA in this picture though. Source: http://www.bio.miami.edu/~cmallery/150/gene/c16x6base-pairs

Base pairs are nucleobases that are building blocks of DNA double helix and even contribute to the folded structure of the DNA because of the positioning of the hydrogen bonds between the bases. There are four base pairs in total: Guanine, Cytosine, Adenine, and Thymine. All of these base pairs only have one opposite that they can bond to in order to create the complementary double stranded DNA. The pairs are Adenine (A) to Thymine (T) and Guanine (G) to Cytosine (C). These pairs are not interchangable and is what makes copying DNA and the polymerase chain reaction possible.




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