BME100 f2013:W1200 Group6 L4

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
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Contents

OUR TEAM

Jenny Chen  Role: Open PCR Machine Engineer
Jenny Chen
Role: Open PCR Machine Engineer
Tracy Lopez Role: Protocol Planner
Tracy Lopez
Role: Protocol Planner
Nayobe Bivins  Role: Research & Development Specialist
Nayobe Bivins
Role: Research & Development Specialist
Alex Bugarin Role: Open PCR Machine Engineer
Alex Bugarin
Role: Open PCR Machine Engineer
Nicholas Kilpatrick Role: Protocol Planner
Nicholas Kilpatrick
Role: Protocol Planner

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

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PCR Machine. Digital image. OpenPCR. N.p., 29 Oct. 2013. Web. <http://openpcr.org/build-it>.

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PCR Machine. Digital image. OpenPCR. N.p., 29 Oct. 2013. Web. <http://openpcr.org/build-it>.

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PCR Machine. Digital image. OpenPCR. N.p., 29 Oct. 2013. Web. <http://openpcr.org/build-it>.



A diagram of the OpenPCR machine with each part labeled.

The OpenPCR machine is a self-built machine capable of accurately maintaining and measuring temperatures for polymerase chain reactions. It comes with an application for the computer that helps in designing protocol and displays a user friendly interface showing the temperature of the thermocycler. There are several key parts that allow this machine to work in an orderly fashion. This includes the heating lid, the heating block, the LCD Screen, the heater, the circuit board, and the fan. The PCR Machine heats up the heating block that contains the test tubes with the samples of DNA with the heater, while the heating lid prevents the heat from escaping. During the denaturing and annealing of the DNA, the DNA continues to replicate causing a large amplification of the the certain strand of DNA. This process is used for gene amplification and can be used to create many copies of the same DNA. This can be used to see if there are infections present or for finding gene variants that can show whether or not a patient is easily susceptible to certain diseases.


Experimenting With the Connections

Unplugging the LCD wires from the circuit board caused the display screen on the machine to turn off, resulting in the PCR Machine not being able to function and making the data unreadable.

Unplugging the white wire that connects the circuit board to the heating block caused the temperature on the screen to decrease from 60°C to -40°C. This wire must be the temperature regulator and without this essential part the machine can not read temperatures correctly.


Test Run

This machine was first tested 10/23/13. As the machine went through the cycles, the temperature changed according to the numbers that were inputed into the computer. The temperature on the computer screen was the same as the one on the PCR Machine throughout all the cycles. Overall the machine ran smoothly and completed the 25 test run cycles giving it a passing mark.




Protocols

Thermal Cycler Program


DNA Sample Set-up

Positive control "PC" Patient 1 ID: 47569 "1" Patient 1 ID: 47569 "2" Patient 1 ID: 47569 "3"
Negative control "NC" Patient 2 ID: 67318 "A" Patient 2 ID: 67318 "B" Patient 2 ID: 67318 "C"



We will be testing 2 patients using PCR. Patient #1 is 47569 and patient #2 is 67318.
Test tubes for patient 1 will be labeled: 1, 2 and 3.
Test tubes for patient 2 will be labeled: A,B and C.

Each patient will have 3 test tubes.
There will also be a positive control and a negative control.
These test tubes are labeled PC and NC respectively.

DNA Sample Set-up Procedure

Step 1: Label all PCR micro tubules according to above designated labels
Step 2: Micro-pipette 50 µL of PCR reaction mix into each tubule
Step 3: Micro-pipette 50 µL of designated DNA primer mix to designated tubules


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We will use the following heating and cooling protocol:

The lid will be heated to 100°C.
We will use an initial step at 95°C for 3 minutes.
We will run 35 cycles to denature, anneal and extend.

It will denature at 95°C for 30 seconds.
It will anneal at 57°C for 30 seconds.
It will extend at 72°C for 30 seconds.

There is a final step at 72°C for 3 minutes.
The final hold will occur at 4°C.



PCR Reaction Mix

  • 50 µL of each; Taq DNA Polymerase, MgCl2 and dNTP's


DNA/ primer mix

  • 50 µL of different template DNA in 8 tubes
  • all eight test tubes will have the same forward and reverse primers




Research and Development

PCR - The Underlying Technology


PCR Components and their Function
The polymerase chain reaction or widely known as the PCR technique has a number of uses. One of which is that it can amplify any target sequence of a single DNA molecule in just a matter of a few hours. Sources of the DNA target sequence can come from: hair strands, blood, or fingerprints at a crime scene investigation; an individual who is afflicted with a virus-causing infection such as AIDS or a bacteria-causing disease like pneumonia; or a preserved thousand-year-old organism. Furthermore, the PCR technique works by mixing the key ingredients in a laboratory instrument called a PCR tube,then the PCR tube and its contents are exposed to repeated cycles of heating and cooling. The main components of a PCR technique include a DNA template,primers, Taq polymerase, magnesium chloride (MgCl2), and deoxyribonucleotides (dNTP’s).


The DNA template contains the target DNA sequence that a researcher wants to amplify. Abovementioned, the DNA sequence can be from an individual, animal, plant, or microorganism. Primers are tiny segments of DNA that bind to a specific site (i.e. on either end of the single-stranded DNA) of the target DNA sequence to initiate the replication of the target DNA sequence. Two primers are usually used in a PCR experiment so that one primer will attach on the top of the DNA strand while the other primer will attach to the other end. When primers are done binding to the DNA strand, a specific type of enzyme called Taq polymerase is activated. This enzyme which is able to withstand heat helps to synthesize new strands of DNA that are identical to the target DNA sequence. A buffer and cofactor for Taq polymerase called magnesium chloride (MgCl2) is added to the PCR tube to stabilize the DNA strand. Lastly, the four different deoxyribonucleotides adenine (A), thymine (T), cytosine (C), and guanine (G) are basically the building blocks for creating new strands of DNA.


Base Pairing
The four dNTP's (or deoxyribonucleotides) used for DNA synthesis and PCR are adenine (A), thymine (T), guanine (G), and cytosine (C). The base pairing of deoxyribonucleotides are always complementary: adenine (A) will bind to thymine (T) and thymine (T) will bind with adenine (A), while guanine (G) will only bind with cytosine (C) and cytosine (C) will bind with guanine (G). For example, if the section of the DNA strand has a sequence like A T T G C C A G, then the strand bound to it would be T A A C G G T C. Consequently, the four deoxyribonucleotides of the DNA template instructs the order of the deoxyribonucleotides being built on the new DNA strand.


Steps of Thermal Cycling
The PCR tube and its contents are positioned in a laboratory instrument that is called a PCR machine or sometimes a thermal cycler. This machine helps facilitate raising and lowering the temperature of a solution to yield a chemical reaction (i.e. the contents in the PCR tube). The temperature of the samples are raised to 95°C for three minutes to initially activate taq polymerase. Then the first official step is to maintain the temperature at 95°C for 30 seconds so that the double helix of the DNA will split into two single stranded fragments. The second step is the annealing. During this process, the temperature is lowered to 57°C for 30 seconds so that hydrogen bonds can be formed between the primers with the single-stranded DNA template. Next is the extension of the DNA wherein the temperature is raised to 72°C; 72°C is the optimal temperature for DNA synthesis. During this step the activated taq polymerase binds beside the primers to continue the synthesis of a newly DNA strand by pairing complementary deoxyribonucleotides to the DNA template. Finally, the solution remains at 72°C for three additional minutes, giving the DNA polymerase enough time to add additional base-pairings before the temperature is lowered to 4°C in order to give the PCR tube time to cool down before starting the next cycle.

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