BME103 s2013:T900 Group4

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(OUR TEAM)
(Research and Development)
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==Research and Development==
==Research and Development==
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Basic DNA components:
+
Mixture components:
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DNA Template <br>
+
DNA Template (Sample DNA)<br>
DNA Polymerase<br>
DNA Polymerase<br>
DNA Primers<br>
DNA Primers<br>
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Nucleotides<br>
+
Free Nucleotides<br>
Procedure:<br>
Procedure:<br>
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First, the components are combined and heated. Once the reaction is heated to a temperature of 95 degrees Celsius, the hydrogen bonds between the DNA strands break and create two separate DNA sequences. After that, leave it to cool down to a temperature of around 55 degrees Celsius.  This allow the DNA polymerase is going the bind the DNA sequence with the DNA Primer with hydrogen bonding. Once, the DNA polymerase is bound, it is going to take some of the free nucleotides from the surrounding area and complete the DNA strand. The result is a two separate DNA sequences, with two DNA strand identical to the first one. Repeatedly doing the steps multiple times and the result will give a total of 4 double DNA strands, and so on...
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First the DNA sample, polymerase, primers, and nucleotides are combined in a small test tube. Repeat samples are advised to make sure that the results are consistent. When the test tube is placed in a PCR machine, the mixture is heated to 95 degrees Celsius for 30 seconds. This high heat breaks apart complementary strands of DNA, exposing the nucleotides. The machine then rapidly cools to 55 degrees Celsius for 30 seconds, allowing the DNA primers to bind to the exposed sites. The DNA primers can only bind to the specific site they are complementary to, which allows researchers to target specific DNA sequences as discussed below in the cancer detection section. The mixture is then heated up to 82 degrees Celsius for 30 seconds. At this temperature, DNA polymerase binds to the primers and builds a new segment of DNA with the free nucleotides in the mixture. This new strand of DNA is complementary to the original. So, if there was only one strand of DNA in the mixture originally, now there are two (since both halves have been replicated). The mixture is then reheated to 95 degrees and the cycle continues, each time doubling the amount of DNA in the mixture. This process usually continues for an hour or so, until there are billions of copies of DNA. This whole process is referred to as DNA amplification.
'''Specific Cancer Marker Detection - The Underlying Technology'''<br>
'''Specific Cancer Marker Detection - The Underlying Technology'''<br>

Revision as of 02:44, 26 March 2013

BME 103 Spring 2013 Home
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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Contents

OUR TEAM

Name: Kinjal Ahir Role:Experimental Protocol Planner
Name: Kinjal Ahir Role:Experimental Protocol Planner
Name: Zach Young, Role:Initial Machine Testing
Name: Zach Young, Role:Initial Machine Testing
Name: Amelia LaxResearch and Development
Name: Amelia Lax
Research and Development
Name: Anna Essex Role:Initial Machine Testing
Name: Anna Essex Role:Initial Machine Testing
Name: student Tuan Phan Role:Research and Development
Name: student Tuan Phan Role:Research and Development
Name: studentRole(s)
Name: student
Role(s)

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
(Add image of the full OpenPCR machine here, from the Week 9 exercise. Write a paragraph description for visitors who have no idea what this is) Here's the OpenPCR machine. Made up of thin pieces of wood, the case contains the hardware of the machine. With another smaller wood case on top of the main case containing the loading bay which acts as a lid. Small test vials can be placed into holders here in the bay to be heated by PCR machine. Inside the main body of the machine sits the hardware similar to a personal computer. A processor unit connects to the heating hardware as well as the power supply and led display board. Above the power supply lies the heatsink with a fan attached facing vents in the case allowing for efficient thermal control inside the machine to prevent overheating.


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

Thermal Cycler Program

DNA Sample, 50 μL each: patient ID's from the UnderGrad Teaching Assistant(UGTA).

Positive control Patient 1 Patient 1 Patient1
cancer DNA template ID: 85158 ID: 85158 ID: 85158
Replicate 1 Replicate 2 Replicate 3
Tube label: A+ Tube label: B1 Tube label: C1 Tube label: D1
Negative Control: no Patient 2 Patient 2 Patient 2
DNA template ID: 17818 ID: 17818 ID: 17818
Replicate 1 Replicate 2 Replicate 3
Tube label: A- Tube label: B2 Tube label: C2 Tube label: D2

DNA Sample Set-up Procedure

  1. Step 1: Student were Provided with 8 tubes of 50 μL PCR reaction mix, 8 tubes of 50 μL diluted template + primers, and disposable transfer pipettes.
  2. Step 2: Protocol planner labeled the test tube that is provided above.
  3. Step 3: 50μL of each DNA sample Mix in to test tube.
  4. Step 4: Protocol planner set up the DNA and ran the PCR reactions.


PCR Reaction Mix

  • In the PCR reaction mix there is Mix contains Taq DNA polymerase, MgCl2, dNTP's, forward primer, reverse primer.

DNA/ primer mix

  • In the DNA/ primer mix there is patient's DNA.






Research and Development

Mixture components:

DNA Template (Sample DNA)
DNA Polymerase
DNA Primers
Free Nucleotides

Procedure:
First the DNA sample, polymerase, primers, and nucleotides are combined in a small test tube. Repeat samples are advised to make sure that the results are consistent. When the test tube is placed in a PCR machine, the mixture is heated to 95 degrees Celsius for 30 seconds. This high heat breaks apart complementary strands of DNA, exposing the nucleotides. The machine then rapidly cools to 55 degrees Celsius for 30 seconds, allowing the DNA primers to bind to the exposed sites. The DNA primers can only bind to the specific site they are complementary to, which allows researchers to target specific DNA sequences as discussed below in the cancer detection section. The mixture is then heated up to 82 degrees Celsius for 30 seconds. At this temperature, DNA polymerase binds to the primers and builds a new segment of DNA with the free nucleotides in the mixture. This new strand of DNA is complementary to the original. So, if there was only one strand of DNA in the mixture originally, now there are two (since both halves have been replicated). The mixture is then reheated to 95 degrees and the cycle continues, each time doubling the amount of DNA in the mixture. This process usually continues for an hour or so, until there are billions of copies of DNA. This whole process is referred to as DNA amplification.

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.)





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