BME100 f2013:W900 Group8 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: Samuel Butler Role(s) Research and Development
Name: Samuel Butler
Role(s)
Research and Development
Name: Kendall HickieRole(s) Protocol Planning
Name: Kendall Hickie
Role(s)
Protocol Planning
Name: Ashley IvanyRole(s) Research and Development
Name: Ashley Ivany
Role(s)
Research and Development
Name: Connor MccoyRole(s) OpenPCR- Maching Testing
Name: Connor Mccoy
Role(s)
OpenPCR- Maching Testing
Name: Kavin WatsonRole(s): OpenPCR- Machine Testing
Name:
Kavin Watson
Role(s):
OpenPCR- Machine Testing

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

Image:Openpcrdenaturing.jpg

Figure 1: The OpenPCR Machine LCD display during DNA denaturing phase. (Image from blog.arduino.cc used under creative commons public license)

Experimenting With the Connections

When part 3 was unplugged (Backlit LCD Display) from part 6 (The "Brain" Board), the machine's LCD display remained backlit, but ceased to display information of any kind.

When the white wires that connects part 6(The "Brain" Board) to part 2 (The Heat Block) were unplugged, the machine's part 3 (Backlit LCD Display) displayed a block temperature of -40 degrees C rather than the usual 27 degrees C.

Test Run

The test run of the OpenPCR machine began on the 23rd of October 2013 at 10:06 AM and ended at 11:16 AM with 25 cycles completed. The machine had only 31 minutes left to make a complete run. Our experience with the OpenPCR Machine was quite pleasant because we never ran into any hitches during the test run.

Protocols

Thermal Cycler Program

  • The thermal cycler program that will be used is the Open PRC Program.


DNA Sample Set-up

Positive Control: Cancer DNA Template Tube Label: + Patient 1 ID: 10072 Replicate 1 Tube Label: 1APatient 1 ID: 10072 Replicate 2 Tube Label: 1BPatient 1 ID: 10072 Replicate 3 Tube Label: 1C
Negative Control: Cancer DNA Template Tube Label: - Patient 2 ID: 83576 Replicate 1 Tube Label: 2APatient 2 ID: 83576 Replicate 2 Tube Label: 2BPatient 2 ID: 83576 Replicate 3 Tube Label: 2C


DNA Sample Set-up Procedure

  1. Each of the test tubes will be labeled with the proper label indicating which patient and which replication will be in which test tube.
  2. 50μL of PCR reaction will be added to each of the eight test tubes by pipette.
  3. The pipette tips will be disposed of after adding each sample to eliminate the chance of cross contaminating samples.
  4. 50μL of DNA primer mix will then be added to each of the 8 test tubes that already contain the reaction mix.
  5. The pipette tips will be disposed of after adding each sample to eliminate the chance of cross-contaminating samples.
  6. Each test tube will then be stored properly.

PCR Reaction Mix

  • The PCR reaction mix contains Taq DNA polymerase, MgCl2, and dNTP's.

DNA/ primer mix

  • The DNA primer mix contains a different template of DNA and have the same forward primer and reverse primer.




Research and Development

PCR - The Underlying Technology


The following represent each function for the componets of a PCR reaction
Template DNA: The template DNA contains the target sequence
Primers: Short pieces of single stranded DNA which are complementary to the DNA. The polymerase then begins synthesizing new DNA from the end of the primer.
Taq Polymerase: A type of enzyme that synthesizes new strands of DNA complementary to the target sequence.
Magnesium Chloride (MgCl2): Acts as a catalyst during the reaction to speed up the heating and cooling process.
Deoxyribonucleotides:Single units of the bases: A, T, C, G. These are building blocks for new DNA strands.


The following happends during each step of thermal cycling
Initial Step: (95 degrees C) for 3 minutes: Reduces non-specific amplification during the inital set up stages of PCR. The DNA sample is then placed and ready for amplification
Denature at 95 degrees C for 30 seconds: The first regular cycling event. This causes DNA meling of the DNA tempalte by disrupting hydrogen bonds between complementary bases.
Anneal at 57 degrees C for 30 seconds: Reaction temperature is lowered, allowing the attachment of primers to the DNA strands.
Extend at 72 degrees C for 30 seconds: The DNA polymerase now synthesizes new DNA strands complementary to the original DNA strand.
Final Step at 72 degrees C for 3 minutes: This ensures the remaining single stranded DNA is fully extended.
Final Hold at 4 degrees C: Allows finilization of process, this could be employed for short term storage of the reaction.

Base Pairing:
Adenine(A):T
Thymine(T):A
Cytosine(C):G
Guanine(G):C

Image:PCR_STORY_BOARD.png
Figure 2: Illustration of how Polyamerase Chain Reaction (PCR) and DNA amplification works. Image Source: DNA Learning Center Website.





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