BME100 f2013:W1200 Group1 L4

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(Initial Machine Testing)
Current revision (15:36, 30 October 2013) (view source)
(Protocols)
 
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{| style="wikitable" width="700px"
{| style="wikitable" width="700px"
|- valign="top"
|- valign="top"
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| [[Image:BME103student.jpg|100px|thumb|Name: Chloe Kwon<br>Role: Research and Development]]
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| [[Image:group1doctor.jpg.jpg|150px|thumb|Name: Chloe Kwon<br>Role: Research and Development]]
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| [[Image:BME103student.jpg|100px|thumb|Name: Nima Afzalian<br>Role: Open PCR Machine Testing]]
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| [[Image:NIMAt.jpg|200px|thumb|Name: Nima Afzalian<br>Role: Open PCR Machine Testing]]
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| [[Image:BME103student.jpg|100px|thumb|Name: Casey Weinstein<br>Role: Open PCR Machine Testing]]
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| [[Image:group1sparky.jpg|200px|thumb|Name: Casey Weinstein<br>Role: Open PCR Machine Testing]]
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| [[Image:BME103student.jpg|100px|thumb|Name: Jeff Miles<br>Role: Protocol Planning]]
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| [[Image:group1pitchfork.jpg|300px|thumb|Name: Jeff Miles<br>Role: Protocol Planning]]
|}
|}
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   [[Image:Group1PCRsetup.jpg|300px]]
   [[Image:Group1PCRsetup.jpg|300px]]
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The image shown above is of a device called an OpenPCR machine. An OpenPCR machine is a low cost, yet highly accurate thermocycler capable of performing polyermase chain reactions that amplifies a specific target DNA segment while maintaining extreme differences in temperature change. After going through many cycles of heating and cooling, the OpenPCR machine replicates a single strand of DNA into many copies. As seen in the image, the OpenPCR machine is connected to a computer using a USB cable. The computer uses an OpenPCR program that allows the number of cycles and each step and desired temperature and time to be adjusted accordingly. Thus, the OpenPCR machine begins the process according to the information inserted into the program.
+
The image shown above is of a device called an OpenPCR machine. An OpenPCR machine is a low cost, yet highly accurate thermocycler capable of performing polymerase chain reactions that amplifies a specific target DNA segment while maintaining extreme differences in temperature change. After going through many cycles of heating and cooling, the OpenPCR machine replicates a single strand of DNA into many copies. As seen in the image, the OpenPCR machine is connected to a computer using a USB cable. The computer uses an OpenPCR program that allows the number of cycles and each step and desired temperature and time to be adjusted accordingly. Thus, the OpenPCR machine begins the process according to the information inserted into the program.
[[Image:group1PCRinside.jpg|400px]]
[[Image:group1PCRinside.jpg|400px]]
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'''Test Run'''
'''Test Run'''
<br>
<br>
-
Our group's PCR machine was tested on October 23. The test began at 1:08 pm. After 30 minutes, 8 cycles were completed.  
+
Our group's PCR machine was tested on October 23. The test began at 1:08 pm. After 30 minutes, 8 cycles were completed. The OpenPCR machine appeared to be running smoothly.
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'''Initial Step'''
'''Initial Step'''
-
95°C for 3 minutes: ''The initial Template DNA strand separates''
+
95°C for 3 minutes: ''The initial Template DNA strand separates.''
'''Denature'''
'''Denature'''
-
95°C for 30 seconds: ''The DNA double helix separates, creating two single-stranded DNA molecules''
+
95°C for 30 seconds: ''The DNA double helix separates, creating two single-stranded DNA molecules.''
'''Anneal'''
'''Anneal'''
-
57°C for 30 seconds: ''The two primers attach to opposite ends of the top and bottom strand of the target DNA segment''
+
57°C for 30 seconds: ''The two primers attach to opposite ends of the top and bottom strand of the target DNA segment.''
'''Extend'''
'''Extend'''
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4°C: ''The PCR reaction ends.''
4°C: ''The PCR reaction ends.''
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[[Image:group1PCRprogram.jpg]]
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[[Image:group1PCRprogram.jpg|300px|]]
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| Tube A: Positive Control: cancer DNA template || Tube B: Patient 1 ID-16946 Replicate 1 || Tube C: Patient 1 ID-16946 Replicate 2 || Tube D: Patient 1 ID-16946 Replicate 3
| Tube A: Positive Control: cancer DNA template || Tube B: Patient 1 ID-16946 Replicate 1 || Tube C: Patient 1 ID-16946 Replicate 2 || Tube D: Patient 1 ID-16946 Replicate 3
|-
|-
-
| Tube E: Negative Control: non-cancer DNA template || Tube F: Patient 2 ID-46296 Replicate 1 || Tube G: Patient 2 ID-46296 Replicate 2 || Tube G: Patient 2 ID-46296 Replicate 3
+
| Tube E: Negative Control: non-cancer DNA template || Tube F: Patient 2 ID-46296 Replicate 1 || Tube G: Patient 2 ID-46296 Replicate 2 || Tube H: Patient 2 ID-46296 Replicate 3
|}
|}
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'''PCR - The Underlying Technology'''<br>
'''PCR - The Underlying Technology'''<br>
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(Add a write-up, essay-style, organized into paragrpahs with descriptive headers, based on the Q&A's from Section three of your worksheet)<br>
 
'''What is the function of each component of a PCR reaction?'''
'''What is the function of each component of a PCR reaction?'''
-
   
 
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(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.)
+
Template DNA is the sample DNA that contains the target DNA segment. Two primers are designed to match to the segment of desired DNA to copy. One primer attaches to the top strand at one end of the target DNA segment and the other primer attaches to the bottom strand at the opposite end. Taq Polymerase is an enzyme that synthesizes new strands of DNA complimentary to the target strand. It can generate new strands of DNA using a DNA template and primers. It is heat resistant. Magnesium Chloride is how magnesium is added to the PCR reaction. It acts like a catalyst. It is not consumed in the reaction but is a necessary component. The more Magnesium Chloride is added, the faster the reaction goes. Deoxyribonucleotides provide the energy and nucleotides for the synthesis of DNA. The nucleotides include Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). They are collected by the Taq Polymerase and used in complementary sequencing of the template DNA strand.
 +
'''What happens during each step of thermal cycling?'''
 +
The initial step of thermal cycling takes place at 95°C for 3 minutes. In this step, the single-stranded template DNA separates. The next step also takes place at 95°C but for 30 seconds and involves the denature of the double-stranded DNA. Each double-stranded DNA separates due to the high heat into single-stranded DNA segments. Next, anneal occurs at 57°C for 30 seconds. The thermal cycler cools down. When this happens, the single-stranded DNA segments try to pair up. One of the primers attach to the top strand of DNA at one end while the other primer attaches to the bottom strand on the opposite end of the DNA segment. As the thermal cycler heats back up to 72°C, the step of extending occurs for 30 seconds. The added heat activates DNA polymerase. Taq Polymerase locates a primer attached to the single-stranded DNA segment and adds complementary nucleotides. The final step of thermal cycling occurs at 72°C for 3 minutes. The complementary binding of nucleotides continues until it gets to the end of the strand and falls off. The final hold of the process happens at 4°C. This step ends thermal cycling.
 +
 +
'''Which base sticks to each base listed below?'''
 +
 +
Adenine (A) is paired up with the base Thymine (T). Thymine (T) is paired up with the base Adenine (A). Cytosine (C) is paired up with the base Guanine (G). Guanine is paired up with the base Cytosine (C).
 +
 +
 +
   
 +
The image below correctly displays the thermal cycling process of how primers bind to the cancer DNA template and how Taq polymerase amplifies the DNA.
 +
[[Image:NIMANAINI.PNG|800px|]]
<br><br>
<br><br>

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Contents

OUR TEAM

Name: Chloe KwonRole: Research and Development
Name: Chloe Kwon
Role: Research and Development
Name: Nima AfzalianRole: Open PCR Machine Testing
Name: Nima Afzalian
Role: Open PCR Machine Testing
Name: Casey WeinsteinRole: Open PCR Machine Testing
Name: Casey Weinstein
Role: Open PCR Machine Testing
Name: Jeff MilesRole: Protocol Planning
Name: Jeff Miles
Role: Protocol Planning

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

 

The image shown above is of a device called an OpenPCR machine. An OpenPCR machine is a low cost, yet highly accurate thermocycler capable of performing polymerase chain reactions that amplifies a specific target DNA segment while maintaining extreme differences in temperature change. After going through many cycles of heating and cooling, the OpenPCR machine replicates a single strand of DNA into many copies. As seen in the image, the OpenPCR machine is connected to a computer using a USB cable. The computer uses an OpenPCR program that allows the number of cycles and each step and desired temperature and time to be adjusted accordingly. Thus, the OpenPCR machine begins the process according to the information inserted into the program.

The image shown above is of the inside of the OpenPCR machine. The environmental changes of heating and cooling are controlled by the heater, heating lid, and cooling fan. The data collected is computed and transferred from the circuit board to the LED screen on top of the OpenPCR machine.


Experimenting With the Connections

The PCR machine is heavily dependent on numerous connections and and interconnected parts. This means that, in order for it to work well, everything must be in its proper location and plugged in. This being said, when we unplugged part 3 (the LCD screen) from part 6 (circuit board) of the machine, the connection from the LCD to the circuit board was severed, resulting in the screen turning off. Also, when we unplugged the white wire that connects part 6 (circuit board) to part 2 (core) of the machine, the temperature changed from 26.7 degrees Celsius to -40 degrees Celsius. It is very important that all connections are correct in order to have an effective PCR machine.


Test Run
Our group's PCR machine was tested on October 23. The test began at 1:08 pm. After 30 minutes, 8 cycles were completed. The OpenPCR machine appeared to be running smoothly.




Protocols

Thermal Cycler Program

Initial Step

95°C for 3 minutes: The initial Template DNA strand separates.

Denature

95°C for 30 seconds: The DNA double helix separates, creating two single-stranded DNA molecules.

Anneal

57°C for 30 seconds: The two primers attach to opposite ends of the top and bottom strand of the target DNA segment.

Extend

72°C for 30 seconds: The DNA polymerase activates. Replication of the target DNA segment begins.

Final Step

72°C for 3 minutes: The complementary binding of nucleotides continues until it gets o the end of the DNA strand and falls off.

Final Hold

4°C: The PCR reaction ends.



DNA Sample Set-up

Tube A: Positive Control: cancer DNA template Tube B: Patient 1 ID-16946 Replicate 1 Tube C: Patient 1 ID-16946 Replicate 2 Tube D: Patient 1 ID-16946 Replicate 3
Tube E: Negative Control: non-cancer DNA template Tube F: Patient 2 ID-46296 Replicate 1 Tube G: Patient 2 ID-46296 Replicate 2 Tube H: Patient 2 ID-46296 Replicate 3


DNA Sample Set-up Procedure

  1. Step 1: Fill each reaction tube with 50μL of the PCR reaction mix and label all tubes according to the chart above
  2. Step 2: Add 50μL of DNA sample mix to the corresponding reaction tube (Note: use each disposable pipette tip only once to avoid cross-contamination)
  3. Step 3: Place to reaction tubes into the thermocycler and run the above program


PCR Reaction Mix

The PCR reaction mix will contain 8 tubes. Each tube will contain 50μL of the following:

  • Taq DNA polymerase
  • MgCl2
  • dNTP's


DNA/ primer mix

The DNA primer mix contains 50μL of the following:

  • an extracted sample of the patient's DNA
  • forward primer
  • reverse primer

Research and Development

PCR - The Underlying Technology

What is the function of each component of a PCR reaction?

Template DNA is the sample DNA that contains the target DNA segment. Two primers are designed to match to the segment of desired DNA to copy. One primer attaches to the top strand at one end of the target DNA segment and the other primer attaches to the bottom strand at the opposite end. Taq Polymerase is an enzyme that synthesizes new strands of DNA complimentary to the target strand. It can generate new strands of DNA using a DNA template and primers. It is heat resistant. Magnesium Chloride is how magnesium is added to the PCR reaction. It acts like a catalyst. It is not consumed in the reaction but is a necessary component. The more Magnesium Chloride is added, the faster the reaction goes. Deoxyribonucleotides provide the energy and nucleotides for the synthesis of DNA. The nucleotides include Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). They are collected by the Taq Polymerase and used in complementary sequencing of the template DNA strand.

What happens during each step of thermal cycling?

The initial step of thermal cycling takes place at 95°C for 3 minutes. In this step, the single-stranded template DNA separates. The next step also takes place at 95°C but for 30 seconds and involves the denature of the double-stranded DNA. Each double-stranded DNA separates due to the high heat into single-stranded DNA segments. Next, anneal occurs at 57°C for 30 seconds. The thermal cycler cools down. When this happens, the single-stranded DNA segments try to pair up. One of the primers attach to the top strand of DNA at one end while the other primer attaches to the bottom strand on the opposite end of the DNA segment. As the thermal cycler heats back up to 72°C, the step of extending occurs for 30 seconds. The added heat activates DNA polymerase. Taq Polymerase locates a primer attached to the single-stranded DNA segment and adds complementary nucleotides. The final step of thermal cycling occurs at 72°C for 3 minutes. The complementary binding of nucleotides continues until it gets to the end of the strand and falls off. The final hold of the process happens at 4°C. This step ends thermal cycling.

Which base sticks to each base listed below?

Adenine (A) is paired up with the base Thymine (T). Thymine (T) is paired up with the base Adenine (A). Cytosine (C) is paired up with the base Guanine (G). Guanine is paired up with the base Cytosine (C).


The image below correctly displays the thermal cycling process of how primers bind to the cancer DNA template and how Taq polymerase amplifies the DNA.


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