BME103:T130 Group 14

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#After the DNA has been through the numerous cycles, you will have over thousands of fragments of the same DNA sequences. After the DNA has been through the thermal cycle, mix each DNA sample with the PCR reaction mix (Taq DNA polymerase, MgCl2, dNTP’s, and a forward and reverse primer), using a separate pipette each time to reduce cross-contamination into 8 separate tubes (see '''Table 2''').
#After the DNA has been through the numerous cycles, you will have over thousands of fragments of the same DNA sequences. After the DNA has been through the thermal cycle, mix each DNA sample with the PCR reaction mix (Taq DNA polymerase, MgCl2, dNTP’s, and a forward and reverse primer), using a separate pipette each time to reduce cross-contamination into 8 separate tubes (see '''Table 2''').
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'''Table 1'''
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! scope="col" | '''Sample Descriptions (8 Samples)'''
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| Positive Control: Cancer DNA Template
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| Patient 1: 44231
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| Patient 1: 44231
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| Patient 1: 44231
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| Negative Control: No DNA Template
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| Patient 2: 57447
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|Patient 2: 57447
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|Patient 2: 57447
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'''Table 2'''

Revision as of 15:47, 8 November 2012

BME 103 Fall 2012 Home
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Contents

OUR TEAM

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LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
(Add image of the full OpenPCR machine here, from the Week 3 exercise. Write a paragraph description for visitors who have no idea what this is)


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

The polymerase chain reaction (PCR) is a biochemical machine used in biological chemistry to produce numerous copies of a particular piece of DNA, generating multiple duplicates of DNA sequences. The PCR machine works similar the cycle of DNA replication at the cellular level. The machine consists of four individual steps, initiation, denaturation, annealing, and extension. The initiation step is solely to prepare the DNA samples to be put through the thermal cycler program. During the denaturation step, the DNA strand is split into two separate strands. After, the annealing step is where the DNA primer attaches to the targeted DNA sequence. The primer only attached to a specific site on the strand, not necessarily the entire strand. The purpose of the primer is to mark the beginning and the end of the targeted DNA sequence. Lastly, in the extension step, the DNA polymerase is first activated, which begins to synthesize the DNA primer. This results in two double stranded target DNA sequences. The PCR machine cycles numerous times to amplify the specific sequence. In order to complete the reaction several components are required such as:
-DNA template
- A PCR reaction mix that contains: Taq DNA polymerase, MgCl2, dNTP’s, and a forward and reverse primer
Most PCR methods use thermal cycling, alternating heating and cooling steps. These thermal cycling steps are necessary to separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At the lower temperature, the DNA polymerase to amplify a particular target DNA uses each DNA strand as a template in DNA synthesis. The primers aid discrimination of the DNA to target the specific region for amplification under specific thermal conditions. The stages of the PCR machine are listed below:

  1. Acquire the DNA samples that have been submitted for testing
  2. Run the DNA samples through the thermal cycler program (see Stage 1)
    1. Stage 1 (Initiation): 1 cycle at 95°C for 3 minutes, to separate the DNA strand.
    2. Stage 2 (Denaturation): 35 cycles: first at 95°C for 30 seconds, and then gradually decrease the temperature to approximately 57°C for 30 seconds, and then raise the temperature to approximately 72°C for 30 seconds, so the DNA polymerase can be activated. This is also an example of heat shock, and is effective to initiate the addition of complementary nucleotides onto the DNA strand, which the DNA polymerase does.
    3. Stage 3 (Elongation) : At this step, hold the DNA at 72°C for 3 minutes.The temperature is held here so that the DNA polymerase can copy the strand. Also, this is where 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 began with.
    4. Stage 4: Final hold until the DNA stabilizes at 4°C. At the end of this cycle you will have 8 fragments of the DNA (see Table 2)
  3. After the DNA has been through the numerous cycles, you will have over thousands of fragments of the same DNA sequences. After the DNA has been through the thermal cycle, mix each DNA sample with the PCR reaction mix (Taq DNA polymerase, MgCl2, dNTP’s, and a forward and reverse primer), using a separate pipette each time to reduce cross-contamination into 8 separate tubes (see Table 2).


Reagent Volume
Template DNA ( 20 nanograms) 0.2 microliters
10 micrometers forward primer 1.0 microliters
10 micrometers reverse primer 1.0 microliters
GoTaq Master Mix 50.0 microliters
Distilled Water 47.8 microliters
Total Volume 100.0 microliters

Table 1

Sample Descriptions (8 Samples)
Positive Control: Cancer DNA Template Patient 1: 44231 Patient 1: 44231 Patient 1: 44231
Negative Control: No DNA Template Patient 2: 57447 Patient 2: 57447 Patient 2: 57447

Table 2


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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