BME100 s2014:W Group1 L4

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BME 100 Spring 2014 Home
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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Contents

OUR TEAM

Name: Maria Jose QuezadaRole(s): Protocols
Name: Maria Jose Quezada
Role(s): Protocols
Name: Danielle EldredRole(s): Open PCR and Machine Testing
Name: Danielle Eldred
Role(s): Open PCR and Machine Testing
Name: John TobeyRole(s): Research and Development
Name: John Tobey
Role(s): Research and Development
Name: David KishRole(s): Open PCR and Machine Testing
Name: David Kish
Role(s): Open PCR and Machine Testing
Name: Khalid Al-QahtaniRole(s): Research and Development
Name: Khalid Al-Qahtani
Role(s): Research and Development

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

Open PCR is machine that is designed to amplify a specific strand of DNA through polymerase chain reaction. In order to fulfill its objective, a sample of DNA undergoes specific temperature cycles. Varying the temperature from 4ºC up to 95ºC allows the double stranded DNA to divide itself into two single strands. After DNA is denatured, the machine is cooled so that the primers can bind to the desired location in the single strand of DNA, and so that the enzyme, taq polymerase, can create the complementary strand of each single strand. This process may be repeated as long as there are free nucleotides. As a result, DNA is duplicated and new double stranded DNA sections are formed.


Image:PCR2.jpg

Fig. 1 Outside structure of Open PCR Machine

Image:PCR.jpg

Fig. 2 Inside components of Open PCR machine

Experimenting With the Connections

When we unplugged (part 3) from (part 6) the display was disconnected from the machine and therefore went black.

When we unplugged the white wire that connects (part 6) to (part 2), the temperature reading displayed a default temperature. The white wire transmits the heat pad data to the rest of the machine. Without this, the temperature readings are not accurate.

Test Run

This machine was first tested on March 19, 2014. The machine remained at cycle one for an hour because it was unable to cool down to 57 degrees which is last step. It was noticed that the fan is not working properly. It was concluded that the Open PCR did not work.




Protocols

Thermal Cycler Program

1. Initial Step (Initial Denaturation) Single cycle of three minutes at 95ºC.

2. Middle Step

35 cycles consisting of:

  • Denature cycle: 30 seconds at 95ºC where the double stranded DNA is divided intro two single strands because the high temperature breaks the hydroden bonds that put together the complementary bases in the DNA structure
  • Anneal cycle: 30 seconds at 57ºC where bothe the forward and reverse primers attach to one DNA strand.
  • Extended cycle: 30 seconds at 72ºC where Taq DNA polymerase adds dNTPs in order to extend the primers and replicate the DNA sequence

3. Final Step- Single cycle of three minutes at 72ºC the final extension occurs.

4. Final Holding- Refrigeration at 4ºC, indicating that CPR reaction has ended.


DNA Sample Set-up

Positive Control D+ Patient 1-Replicate 1 Tube Label: 1-1 Patient 1-Replicate 2 Tube Label: 1-2 Patient 1- Replicate 3 Tube Label: 1-3
Negative Control D- Patient 2-Replicate 1 Tube Label: 2-1 Patient 2-Replicate 2 Tube Label: 2-2 Patient 1-Replicate 1 Tube Label: 2-3



DNA Sample Set-up Procedure

  1. Step 1 Prepare the following material:8 empty PCR tubes, reaction mix, template DNA, positive and negative control, disposable pipette tips and 200 μL micropipettor
  2. Step 2 Label the test tubes with permanent black according to the established codes shown in the previous table
  3. Step 2 With the micropipette fill each PCR tube with 50 μL of PCR reaction mix. Remember to discard each disposable tip into the appropriate container after each step in order to reduce cross-contamination.
  4. Step 3 With the micropipette fill each PCR tube with 50 μL of template DNA.
  5. Step 4 According to the labels, add either positive or negative control to each sample.
  6. Step 5 Securely close the lids of the 8 PCR tubes.
  7. Step 6 Place the 8 PCR tubes over the assigned PCR machine and start the machine after one of the TAs checks everything is in order.


PCR Reaction Mix

  • What is in the PCR reaction mix?

The PCR reaction mix contains -Template DNA- Strand of DNA that provides the desired smaller string of nucleotide

-Primers- Primers provide a start point for the desired strand of nucleotide and provide a binding point for Taq

-Taq Polymerase- taq polymerase is a protein that binds to the DNA and helps to add nucleotide to the single of DNA

-Deoxyribonucleotides- The deoxyribonucleotides (bind to the Mg 2+ions) reduce the activity of the enzyme

DNA/ primer mix

  • What is in the DNA/ primer mix?

-The patient's DNA

-Forward Primer

-Reverse Primer




Research and Development

PCR - The Underlying Technology


Function of the Components of a PCR Reaction

In the PCR reaction, there are four main component that are combined to replicate specific genes, or segments of DNA. The template DNA is a strand of DNA that contains the nucleotide sequence that produces the desired protein. Primers are small strands of nucleotides that provide point for the Taq Polymerase to bind to. The primers are designed to bind just before the desired nucleotide strand. Once the protein, Taq polymerase, binds to the primer and it’s complementary bases, the Taq polymerase works to add complementary nucleotides to the single strand of DNA. The Taq polymerase adds deoxyribonucleotides (dNTP’s) to the single strand of DNA. The dNTP’s are made of three parts: nitrogenous base, deoxyribose sugar, and a phosphate group. Depending on it’s structure, a dNTP can be either guanine (G), cytosine (C), adenine (A), or thymine (T). Depending on their order, these four compounds are capable of making innumerable proteins.


PCR Process

The initial step of the polymerase chain reaction is the heating of the components to 95ºC. This step denatures the strand of DNA, with the desired sequence, into two single strands. Next, in the process known as annealing, the mixture is cooled to 57ºC the primers attach to the DNA strands at the desired positions due to their complementary dNTP’s. For the next step, extension, the mixture is heated to 72ºC. This allows for the Taq polymerase to bind to the DNA strands and to build strands of double-helical DNA. This process can be repeated continuously as long as there are enough nucleotides to form DNA. Finally, the mixture is cooled to 4ºC to stabilize the double strands of DNA.

(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.) Image:PCRDNA.jpg




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