BME103 s2013:T900 Group3
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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LAB 1 WRITE-UP
Initial Machine Testing
Experimenting With the Connections
We have to make sure that each piece in the PCR machine working correctly. For example, when we unplugged (part 3)which is LCD Screen from circuit board (part 6),the LSD screen won't show any data set.However, when we unplugged the white wire that connects the circuit boar(part 6) to( part 2) which is the heater, the machine won't give proper temperature.
On March 5,2013,open PCR machine was first tested by our group. After trying several machines and USB cables we setup our actual working machine which is number 3.Connected and uploading the information from the computer to the Open PCR allow us to run our simple diagnostic test, and observed the good result by the end of the test .
Thermal Cycler Program
The PCR tube goes through three different thermal stages:
The temperature heats up to 95 °C for three minuets
A) In stage two, the temperature increases to 95 °C, 205 °F. This boiling temperature separates the DNA helix, making two single-stranded DNA molecules.
B) The PCR tube contains many primer sequences beside DNA strands. However, after separating the DNA double helix, the temperature cools down to 57 °C, about 135 °F. At this temperature, the primers immediately look for and lock onto their targets before the stands can reunite. Because when the heat is 57 °C, the stands tries to find each other to pair up.
C) The temperature heats up to 72 °C, around 163 °F. At this temperature the DNA polymerase becomes active. Then starts adding the complementary nucleotides onto each single DNA strands by attaching both the primer and stands. The DNA polymerase keeps doing this step until it gets to the end of the stand and then falls off.
Temperature is at 72 °C For three minutes.
- These three stages happen in the first cycle, and it keeps repeating it 30 times to get millions copies of DNA stands.
DNA Sample Set-up Procedure
1) 8 tubes contain 50 μL of PCR reaction mix. And another 8 tubes contain DNA Sample/Primer mix.
2) Label the 8 tubes that contain the DNA sample/primer mix (as is shown in the table above).
3) Transfer the whole 50 μL of each PCR reaction mix to the 8-labeled tubes by using transfer pipettes. (used the transfer pipettes once each time.)
4) Now each 8-labeled tube has a mix of PCR reaction and DNA sample/primer. Placed these tubes into PCR machine.
5) Plug in the OCR machine to a computer and set-up the three different stage of the thermal cycler after downloading the "openPCR" application.
6) Click on Run to run the experiment. So the PCR start working.
Taq DNA polymerase.
DNA/ primer mix
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
Polymerase chain reaction is the process of copying DNA. Scientists may use this process to copy and study known cancerous DNA so that they may find resolutions to gene mutations.
To perform the PCR, DNA is obtained, then a specific sequence of nucleotides is chosen and identified to be copied such as the gene mutation and this will be the template DNA. A forward and reverse primer is ordered to complement the template DNA. Adenine only attaches to thymine and cytosine only attaches to guanine. The DNA is mixed with the primers, dNTPs, Taq DNA polymerase, and magnesium chloride. The mix is heated to 95 °C for 30 seconds which unzips the double helix strand in half between the base paired nucleotides. Then it is cooled to 57 °C for 30 seconds which causes primer anneal binding on the ends of the nucleotides. It is heated to 72 °C for 30 seconds, this causes the Taq polymerase which is catalyzed by the magnesium chloride to attach the buoyant dNTP’s to their complementary base pair. This completes one cycle and produces one copy. Performing this procedure 30 times would give approximately 1 million copies of the template DNA.