BME100 f2013:W900 Group11 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: Alvaro RasconOpen PCR testin)
Name: Alvaro Rascon
Open PCR testin)
Name: Timothy Millea  Protocols
Name: Timothy Millea
Protocols
Name: Diana Terreros Research and Development
Name: Diana Terreros
Research and Development
Name: John ShermanOpen PCR testing
Name: John Sherman
Open PCR testing
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
Role(s)

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:Openpcrgroup11.jpg.png

(image credit to google images)

What is a PCR Machine

The Polymerase Chain Reaction machine, or PCR machine, is an important machine that produces copies of DNA and then keep making even more using just one strand of DNA. This machine does this by heating up the segments of DNA to separate them letting the polymers attach and create the other segments then cooling it to bring the segments back together into a complete DNA segment. this is called a Heating cycle that can be used to make many more copies of DNA. It is used for many things like diagnosing certain diseases based on the DNA, or using it as a fingerprint to help catch murderers.


Experimenting With the Connections
When we unplugged (part 3) from (part 6),the display screen turned off. what we unplugged was the power to the display screen so no information could be sent to display on the screen.

When we unplugged the white wire that connects (part 6) to (part 2), the temperature reading that were displayed changed. the white wire controlled the accuracy of the temperature reading and when it was disconnected it did not read the temperatures right.

Test Run

The PCR machine was tested on October 23, 2013. unfortunately the machine did not operate as expected. when the test started with the test run it was supposed to take 7 minutes to warm up but it stayed at that station for about 30-45 min. this clearly showed that the machine was broken at some parts.




Protocols

Thermal Cycler Program

When the OpenPCR machine runs, it begins with the warm-up process. The process that takes 3 min as it warms up to 95 C to denature DNA, and let run for 25 cycles. Repeat, adding PCR reaction mix to each sample, and letting run for 25 cycles. Each cycle is comprised of 30 seconds at 95 C, which separates DNA into separate strands, 30 seconds 57 C for primer attachment, and 72 C to begin action of DNA polymerase. 

DNA Sample Set-up

Positive control: cancer DNA template tube label: Cnr 26317 F Replicate 1 Tube label: 1-1 26317 F Replicate 2 Tube label: 1-2
Negative control: clean DNA template tube label: Cln 63107 M Replicate 1 Tube label: 2-1 63107 M Replicate 2 Tube label: 2-2


DNA Sample Set-up Procedure 1. Label the DNA/primer mix the appropriate labels as detailed above. 2. Add the PCR reaction mix to the DNA/primer mix that will be tested. 3. Initiate PCR machine, and allow cycling to take place.


PCR Reaction Mix

  • The PCR reaction mix contains Tac polymerase and ligase enzymes required to form new DNA, as well as the nucleotides with which the new molecules will form.

DNA/ primer mix

  • The DNA/primer mix contains the sample DNA as well as RNA primers.





Research and Development

PCR - The Underlying Technology

The purpose of Polymerase Chain Reaction is to make a large number of copies of a DNA sequence using primers, which are short pieces of DNA created in a lab to match the template DNA. To complete this process, you need template DNA, which is the sample DNA that contains the target sequence. You will need a Taq polymerase (a DNA polymerase) that is stable in many temperatures. The polymerase is used to synthesize a new DNA strand from the template. It attaches itself near the end of the primer and begins adding nucleotides, which are the building blocks that DNA is made of (A’s, C’s, G’s, and T’s). You will need magnesium chloride, acts as a cofactor or a helper to the activity of the enzymes.

The first step of PCR is denaturation. In this step, the solution is heated to 95 degrees for 30 seconds. During this time the hydrogen bonds in the DNA strand are broken, which allows it to be separated into two strands so that copying may occur. The next step is annealing, in which the solution is cooled down to 57 degrees for 30 seconds. In this step the primers form ionic bonds with the DNA so that the polymerase can start copying. This is where the magnesium chloride comes into play. Because the DNA strands are negatively charged, they need a salt to decrease the repulsion between them. Magnesium does this and allows the two strands to come together and anneal. After this is extenstion. The solution is heated to 72 degrees for 30 seconds because 72 degrees is the ideal temperature for the polymerase. During the 30 seconds, the Taq polymerase adds the deoxyribonucleotides (A’s pair with T’s and C’s pair with G’s), creating two double-stranded DNA strands from the original template DNA. This whole process repeats itself for 30-40 cycles until there are a lot of copies of the original DNA.





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