Difference between revisions of "BME103:T930 Group 3"

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'''Fluorimeter Assembly'''<br>
'''Fluorimeter Assembly'''<br>
[[Image:BME-03 box.jpg|200px|thumb|frame|left|This is the outer surface of the fluorimeter, and exterior view]]
[[Image:BME-03 box.jpg|200px|thumb|frame|right|This is the outer surface of the fluorimeter, and exterior view]]
[[Image:BME-03 box.jpg|200px|Description of image]]
'''Fluorimeter Procedure:'''<br>
'''Fluorimeter Procedure:'''<br>

Revision as of 22:38, 14 November 2012

Owwnotebook icon.png BME 103 Fall 2012 Home
Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
Course Logistics For Instructors
Wiki Editing Help
BME494 Asu logo.png


Rohan Kumar
Experimental Protocol Planner
Kyle Stoneking
Experimental Protocol Planner
Lekha Anantuni
R&D Scientist
Joshua Eger
Machine Engineer
Austin Cuaderno
Machine Engineer


Initial Machine Testing

The Original Design

BME103 Group3 PCR.jpg

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.

Test Run

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.

Sample one ID 43891: 48 Male
Sample two ID 36890: 56 Female

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

Fluorimeter Assembly

This is the outer surface of the fluorimeter, and exterior view

Description of image

Fluorimeter Procedure:
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

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

(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.)


BME-3 Data.png

Sample Integrated Density DNA μg/mL Conclusion
PCR: Negative Control 4528827 1.08 Positive
PCR: Positive Control 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)
  • 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.