BME103:T930 Group 3
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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
The Original Design
When we unplugged the lcd display wire(part 3) from the Arudnio chip(part 6), the screen turned off. Everything on the PCR was working fine expect there was no output on the display. When we unplugged the white wire that connects Arudino chip(part 6) to thermal cycler (part 2), the reading from the screen dropped to -40 degrees Celsius. We disconnected the wire multiple times and each time the screen displayed -40 degrees Celsius.
We ran a test run on 10/25/2012. For this test we placed some empty PCR tubes into the machine and ran a simple test program on the Open PCR software. After the simple test was over we noticed that the display screen on the Open PCR lid matched very closely with what was displayed on our computer screen. The agreement between our computer screen and our PCR display meant that our diagnostic test was a success.
Polymerase Chain Reaction
Polymerase Chain Reaction Procedure:
2.)We labeled 8 empty PCR tubes. For the first sample we labeled the 3 DNA samples 1A, 1B and 1C. For the second sample we labeled the tubes 2A, 2B and 2C. For the positive and negative controls, we labeled the tubes + and - respectively.
3.)Using one pipette per sample, to avoid contamination, we transferred the PCR reaction mix we were given to the PCR tubes.
4.)We then placed the samples in the PCR machine
5.)We set our PCR program to three stages. Stage one: 1 cycle, 95 degree Celsius for 3 minutes. Stage 2: 35 cycles, 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, 72 degrees Celsius. Stage three: 72 degrees Celsius for 3 minutes and then hold at 4 degree Celsius.
Sample one ID 43891: 48 Male
GO Taq DNA Mix
2.)With the permanent marker we also labeled the Eppendrof tubes at the top, we had a total of 10 Eppendrof tubes labeled and 10 pipettes labeled.
3.)We transferred each sample separately into the Eppendorf tubes containing 400 ml of buffer.
4.)Using a specially labeled Eppendorf tube containing SYBR GREEN, with its own pippter, we placed two drops onto the first two center drops.
5.)Then using the sample we placed two drops on top of the SYBR GREEN solution drops
6.)Then we aligned the blue light to pass through the drop.
7.)Then the smartphone operator took a picture with the settings on the phone adjusted to inactive flash, iso to 800, white balance to auto, exposure to the highest setting and contrast to the lowest setting.
8.)This process was repeated for all samples
9.)After picture was taken it was given to the Image J software
Image J Procedure:
2.)Using the menu selection we used, image > color > split channels
3.) This created 3 files
4.)The we clicked menu bar to activate the oval selection.
5.)We drew an oval around our green drop image and then selected analyze > measure.
6.) We then repeated the oval process but for the area above the drop, to get the noise measurement.
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
We added a specific primer that attaches to the cancer portion of the DNA this Primer duplicates only the DNA after the cancer portion. Because of the way DNA is replicated, after several replications we will be left with primarily the cancer portion of the DNA. The way we get the DNA to replicate is by using the open pcr machine. this machine cycles through different temperature that are optimal for the different phases of the DNA replication. We cycled the machine 30 times to be sure we had enough of the cancer DNA present in our solution.