BME103:T130 Group 13

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(Protocols)
(Protocols)
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There are typically many cycles that need to take place in the PCR in order to amplify a patient's DNA.  
There are typically many cycles that need to take place in the PCR in order to amplify a patient's DNA.  
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<br>'''Cycle 1:''' the thermal cycler heats up to 95 degrees Celsius, or 203 degrees Fahrenheit, which is almost at boiling point. At this temperature, DNA double helix separates, creating two single-stranded DNA molecules.  Gradually the temperature begins to cool to 50 degrees Celsius so the primers will attach. Then the temperature is raised to 72 degrees DNA polymerase is activated and locates primers attached to the single-strand DNA, which will then begin to add complementary nucleotides onto the strand. This process continues until it gets to the end of the strand and falls off.
+
<br>'''Cycle 1:''' The PCR master mix contains dNTPs, MgCl2, and reaction buffers at optimal concentrations for effiecient amplication of DNA templates. The thermal cycler heats up to 95 degrees Celsius, or 203 degrees Fahrenheit, which is almost at boiling point. At this temperature, DNA double helix separates, creating two single-stranded DNA molecules.  Gradually the temperature begins to cool to 50 degrees Celsius so the primers will attach. Then the temperature is raised to 72 degrees DNA polymerase is activated and locates primers attached to the single-strand DNA, which will then begin to add complementary nucleotides onto the strand. This process continues until it gets to the end of the strand and falls off.
<br>'''Cycle 2:''' The same three steps occurring in cycle happen in cycle 2. The temperature is raised again to separate the DNA strands, the temperature is lowered so that the primers may attach, and the temperature is raised again slightly to stimulate DNA polymerase to copy the strand.  
<br>'''Cycle 2:''' The same three steps occurring in cycle happen in cycle 2. The temperature is raised again to separate the DNA strands, the temperature is lowered so that the primers may attach, and the temperature is raised again slightly to stimulate DNA polymerase to copy the strand.  
<br>'''Cycle 3:'''the two desired fragments begin to appear—two strands that begin with primer one and end with primer two—and these are the DNA copies of the segment of DNA you’ve targeted. These products will increase (become the majority) as the cycle continues.  
<br>'''Cycle 3:'''the two desired fragments begin to appear—two strands that begin with primer one and end with primer two—and these are the DNA copies of the segment of DNA you’ve targeted. These products will increase (become the majority) as the cycle continues.  
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<br>'''Cycle 5:''' At the end of this cycle, you‘ll have 22 fragments that your target sequence and only ten longer length copies.  
<br>'''Cycle 5:''' At the end of this cycle, you‘ll have 22 fragments that your target sequence and only ten longer length copies.  
<br>'''After 30 cycles''' there are over a billion fragments that contain only your target sequence and only 60 copies of the longer length molecules. You now have a solution of nearly pure target sequence.  
<br>'''After 30 cycles''' there are over a billion fragments that contain only your target sequence and only 60 copies of the longer length molecules. You now have a solution of nearly pure target sequence.  
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Revision as of 18:41, 1 November 2012

BME 103 Fall 2012 Home
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Lab Write-Up 1
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Contents

OUR TEAM

Name: Sudarshan IyerResearch and Development Specialist
Name: Sudarshan Iyer
Research and Development Specialist
Name: Ujwala VakaExperimental Protocol Planner
Name: Ujwala Vaka
Experimental Protocol Planner
Name: Emily HerringExperimental Protocol Planner
Name: Emily Herring
Experimental Protocol Planner
Name: Garrett ReppOpen PCR Machine Engineer
Name: Garrett Repp
Open PCR Machine Engineer
Name: Joseph Del RosarioOpen PCR Machine Engineer
Name: Joseph Del Rosario
Open PCR Machine Engineer

LAB 1 WRITE-UP

The Original Design
Image:Group_13_Thursday_130_PCR.png


Experimenting With the Connections

When we unplugged (part 3) from (part 6), the machine ... (did what? fill in your answer)

When we unplugged the white wire that connects (part 6) to (part 2), the machine ... (did what? fill in your answer)


Test Run

(Write the date you first tested Open PCR and your experience(s) with the machine)




Protocols

Polymerase Chain Reaction

Polymerase Chain Reaction is a technology that amplifies a single piece of DNA. This technology works very similarly to the natural DNA replication cycle. One PCR cycle consists of three basic steps, denaturation, annealing and extension. In the denaturation step, heat (usually about 95 degrees Celsius) is used to separate the DNA into two strands. Then in the annealing step, the temperature is decreased to 50 degrees Celsius and the DNA primer, specific to the target sequence for that organism, anneal to the separated strand of DNA. The primers mark the beginning and the end of the targeted DNA sequence. Finally, the extension step required the temperature to be raised to 72 degrees Celsius so that the DNA polymerase is activated. The DNA polymerase begins synthesis at the DNA primer. This results in two double stranded target DNA sequences. The PCR cycle is repeated many times to amplify the targeted strand.

There are typically many cycles that need to take place in the PCR in order to amplify a patient's DNA.
Cycle 1: The PCR master mix contains dNTPs, MgCl2, and reaction buffers at optimal concentrations for effiecient amplication of DNA templates. The thermal cycler heats up to 95 degrees Celsius, or 203 degrees Fahrenheit, which is almost at boiling point. At this temperature, DNA double helix separates, creating two single-stranded DNA molecules. Gradually the temperature begins to cool to 50 degrees Celsius so the primers will attach. Then the temperature is raised to 72 degrees DNA polymerase is activated and locates primers attached to the single-strand DNA, which will then begin to add complementary nucleotides onto the strand. This process continues until it gets to the end of the strand and falls off.
Cycle 2: The same three steps occurring in cycle happen in cycle 2. The temperature is raised again to separate the DNA strands, the temperature is lowered so that the primers may attach, and the temperature is raised again slightly to stimulate DNA polymerase to copy the strand.
Cycle 3:the two desired fragments begin to appear—two strands that begin with primer one and end with primer two—and these are the DNA copies of the segment of DNA you’ve targeted. These products will increase (become the majority) as the cycle continues.
Cycle 4: At the end of this cycle, you‘ll have 8 fragments that contain only your target sequence.
Cycle 5: At the end of this cycle, you‘ll have 22 fragments that your target sequence and only ten longer length copies.
After 30 cycles there are over a billion fragments that contain only your target sequence and only 60 copies of the longer length molecules. You now have a solution of nearly pure target sequence.


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Flourimeter Measurements

(Add your work from Week 3, Part 2 here)




Research and Development

Specific Cancer Marker Detection - The Underlying Technology

(Add a write-up of the information discussed in Week 3's class)

(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 OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)




Results

(Your group will add the results of your Fluorimeter measurements from Week 4 here)


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