Experimental Protocol Planner
Experimental Protocol Planner
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
The OpenPCR is a machine that is used to replicate DNA in order to amplify a specific gene. This machine primarily uses changes in temperature and various enzymes to facilitate the replication process multiple times. If the heating lid did not work, then DNA replication would not be possible. The heating lid covers the thermal cycler and holds the DNA down tight and fluctuates in temperature. The thermal cycler is where the samples are place and varies at set temperatures and times. The temperature change is crucial to the replication of DNA because only at certain temperatures dose the DNA properly replicate. The LCD display outputs the temperature of the thermal cycler. The power supply connects to an external power source.
Experimenting With the Connections
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
We have been given 3 sets of samples of replicate DNA from two patients, to test for cancer makers. We labeled each sample carefully as to not cross contaminate the samples. We also used one positive control sample and one negative control, which contained no DNA,to give us a total of 8 samples. We mixed the samples together with Taq DNA polymerase, MgCl2, dNTP'S, forward primer and reverse primer. We used the PCR machine to replicate the DNA. After the PCR had finished replication, drops of the samples, mixed with syber green, were then placed in a fluorimeter. We used a Samsung Galaxy Nexus smartphone to take pictures of each drop. We then used image j to analyze the drops.
Polymerase Chain Reaction Procedure:
1.)We received 3 replicate DNA samples each from two patients and One positive control and negative control sample for a total of 8 samples. The samples we were given were already in their PCR reaction mix form. This mix contained Taq DNA polymerase, MgCl2, dNTP's, forward primer and reverse primer. Each sample was 50 micro liters.
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.
The Patient Information
| Patient Identification Number
GO Taq DNA Mix
| Reagent || Volume
| Template DNA (20 ng) || 0.2 μL
| 10 μM forward primer ||1.0 μL
| 10 μM reverse primer || 1.0 μL
| GoTaq master mix || 50.0 μL
| dH2O || 47.8 μL
| Total Volume || 100.0 μL
1.) The first picture is the extertor of the fuorimeter.
2.) The second picture shows the interior. The smartphone camera is angled to have a view of the slide. During actual use the slide platform was sightly elevated with plates for a better view of the drop.
3.) The third picture shows the slide in view of the camera. During actual use the settings were changed and the lid was closed for optimal accuracy.
4.) The fourth picture is an areial view of the what the slide setup looks like. The water drop is nested in place.
1.) Using permanent marker we numbered the transfer pipette at the bulb, so its only used for one sample
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
1.) The first image is the positive control sample before ImageJ analysis.
2.) The second image is the negative control sample before ImageJ analysis.
Image J Procedure:
1.)Using the menu selection we used, analyze> set measurements and chose area integrated density and mean grey value
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.
| Sample || Integrated Density || DNA μg/mL || Conclusion
| PCR: Negative Control || 4528827 || 1.08 || Positive
| PCR: Positive Control (Calf Thymus) || 8351858 || 2 || Positive
| PCR: Patient 1 ID 43891, rep 1 || 5283304 || 1.26 || Positive
| PCR: Patient 1 ID 43891, rep 2 || 4376083 || 1.05 || Positive(borderline)
| PCR: Patient 1 ID 43891, rep 3 || 8144751 || 1.95 || Positive
| PCR: Patient 2 ID 36890, rep 1 || 4190775 || 1 || Negative(borderline)
| PCR: Patient 2 ID 36890, rep 2 || 5721560 || 1.37 || Positive
| PCR: Patient 2 ID 36890, rep 3 || 5611447 || 1.34 || Positive
- Sample = Sample is the DNA sample we are analyzing
- Integrated Density = The integrated density is similar to a numerical representation of the measure light in a given area. The integrated density in the above table is found by taking the integrated density of the sample and subtracting the background noise from that
- DNA μg/mL = 2 * ( integrated density of sample / integrated density of positive control(calf thymus))
- Conclusion = If the DNA μg/mL is greater than 1 than the sample has indeed replicated. If the sample is less than 1 than it has not shown exponential replication.