BME100 s2014:T Group15 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: Ariana L. Shults
Name: Ariana L. Shults
Name: Samantha J Hatley
Name: Samantha J Hatley
Name: Megan Mendieta
Name: Megan Mendieta
Name: Heston Scott Hamilton
Name: Heston Scott Hamilton
Name: Mohammed A. Tamim
Name: Mohammed A. Tamim
Name: Brenda Cardenas
Name: Brenda Cardenas

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
(Add image of the full OpenPCR machine here, from the Week 9 exercise. Write a paragraph description for visitors who have no idea what this is)
Image:300px-Labeled_OpenPCR.pngImage:Openpcr1.png

An OpenPCR machine is an open-source, hackable, DIY PCR machine. PCR stands for Polymerase Chain Reaction, and it's an important tool for just about any type of modern molecular biology. It works by amplifying a specific part of a strain of DNA in order to study that section more carefully.

Experimenting With the Connections

When we unplugged (part 3) from (part 6), the machine's LCD screen turned off.

When we unplugged the white wire that connects (part 6) to (part 2), the machine the temperature displayed on the LCD screen dropped from 25 degrees C to -40 degrees C.


Test Run

(Write the date you first tested Open PCR and your experience(s) with the machine)
Date: March 20, 2014

We opened the OpenPCR program, added a new experiment, and entered the following 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

We then placed the empty tubes in the OpenPCR machine, turned the knob on the top until there was some resistance which indicated the top was touching the tubes. Next we turned the machine on and pressed the start button on the OpenPCR program. The LCD screen displayed which run it was on and which step within that run. Our OpenPCR machine was a pass, meaning it ran with no problems.



Protocols

Thermal Cycler Program


DNA Sample Set-up

row 1 cell 1 - (+) row 1 cell 2 - (P11) row 1 cell 3 - (P12) row 1 cell 4 - (P13)
row 2 cell 1 - (-) row 2 cell 2 - (P21) row 2 cell 3 - (P22) row 2 cell 4 - (P23)


DNA Sample Set-up Procedure

  1. Step 1- Set up 8 PCR tubes stably in a container of ice (by cutting the tubes into 2 sections of 4), as well as the tubes for the DNA/primer mix and PCR reaction mix. Label the the empty tubes.
  2. Step 2- Using the micro pipet, attach a tip and transfer 50 micro liters of PCR reaction mix and 50 micro liters of the positive control into an empty tube. Make sure to replace the tip each time a mix is transferred.
  3. Step 3- Repeat this process for the negative control.
  4. Step 3- Repeat this process for 3 patient 1 replicates.
  5. Step 4- Repeat this process for 3 replicates of patient 2.
  6. Step 5- Make sure the tubes are closed tightly and place all of the mixed tubes in the OpenPCR Machine.
  7. Step 6- Start the Open PCR Software and enter given values for the heating/cooling protocol specified by the instructor (with the help of a Lab TA).


PCR Reaction Mix

  • What is in the PCR reaction mix?

The mix contains taq DNA polymarase, MgCl2, and dNtp's.


DNA/ primer mix

  • What is in the DNA/ primer mix?

Each mix contains a different template DNA. All tubes have the same foward primer and reverse primer.





Research and Development

PCR - The Underlying Technology

(Add a write-up, essay-style, organized into paragraphs with descriptive headers, based on the Q&A's from Section three of your worksheet)
Functional Components:

DNA is analyzed with PCR reactions with the help of four main components: Template DNA, Primers, Taq Polymerase, and Deoxyribonucleotides (dNTP's). The template DNA is the original strand of DNA, and contains the section that is to be replicated via PCR. The primers attach to the template strand in two places in order to indicate to the DNA Polymerase where to begin and end replication. Taq polymerase handles the replication of the indicated strand by adding on a complimentary set of bases, which are the dNTP's, in between the primers.

PCR Process:

In order to replicate the strand of DNA via PCR, first the template strand is heated to 95 degrees Celsius for 3 minutes in order to begin denaturing. The denaturing continues at 95 degrees for 30 seconds, at which point the template DNA strands are fully separated. The annealing process then begins at 57 degrees Celsius for 30 seconds, which allows the primers to attach to the complementary strands of DNA. This process is extended at 72 degrees Celsius for 30 seconds, which is when the Taq polymerase binds to the primers. The final step is held at 72 degrees for 3 minutes, and the Taq polymerase attaches the nucleotides to create a complementary strand of the copied template DNA.

(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the PCR video/ tutorial might be useful. Be sure to credit the sources if you borrow images.)





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