BME103:T130 Group 7

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Research and Development)
(OUR TEAM)
Line 17: Line 17:
| [[Image:bananafran.jpg|100px|thumb|Name: Frances Marrett<br>Experimental Protocol Planner]]
| [[Image:bananafran.jpg|100px|thumb|Name: Frances Marrett<br>Experimental Protocol Planner]]
| [[Image:cglass.jpg|100px|thumb|Name: Chris Glass<br>Open PCR Machine Engineer<br>[http://openwetware.org/wiki/User:Christopher_M._Glass]]]
| [[Image:cglass.jpg|100px|thumb|Name: Chris Glass<br>Open PCR Machine Engineer<br>[http://openwetware.org/wiki/User:Christopher_M._Glass]]]
-
| [[Image:moose.jpg|100px|thumb|Name: Ryan Frantz<br>Open PCR Machine Engineer]]
+
| [[Image:moose.jpg|100px|thumb|Name: Ryan Frantz<br>Open PCR Machine Engineer<br>[http://openwetware.org/wiki/User:Ryan_T._Frantz]]]
| [[Image:puffer.jpg|100px|thumb|Name: Cenric Nigbur<br>Ghost/ TBD]]
| [[Image:puffer.jpg|100px|thumb|Name: Cenric Nigbur<br>Ghost/ TBD]]
|}
|}

Revision as of 18:54, 12 November 2012

BME 103 Fall 2012 Home
People
Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
Course Logistics For Instructors
Photos
Wiki Editing Help
Image:BME494_Asu_logo.png

Contents

OUR TEAM

Name: Emily ThompsonResearch & Development Scientist
Name: Emily Thompson
Research & Development Scientist
Name: Vivian BenjesExperimental Protocol Planner
Name: Vivian Benjes
Experimental Protocol Planner
Name: Frances MarrettExperimental Protocol Planner
Name: Frances Marrett
Experimental Protocol Planner
Name: Chris GlassOpen PCR Machine Engineer[1]
Name: Chris Glass
Open PCR Machine Engineer
[1]
Name: Ryan FrantzOpen PCR Machine Engineer[2]
Name: Ryan Frantz
Open PCR Machine Engineer
[2]
Name: Cenric NigburGhost/ TBD
Name: Cenric Nigbur
Ghost/ TBD

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
This is a PCR machine (model: Open PCR 1 Jankowski and Perfetto). This machine is capable of replicating DNA through a series of temperature changes over timed cycles.

Image:PCR Machine.png


Experimenting With the Connections

When the PCB board was unplugged from the Open PCR circuit board, the machine's LED display ceased to function.

When the white wire that connects the Open PCR circuit board to 16 tube PCR block was unplugged, the machine's LED display read -40 Celsius indicating that the temperature could not be read.


Test Run

October 18,2012; The test program labeled "Simple Test", was performed to ensure proper function of the Open PCR machine. It ran through two cycles at a full range of temperatures, no DNA was present for the test run. The test performed without any flaws.




Protocols

Polymerase Chain Reaction

To amplify samples of DNA, the OpenPCR machine was used to perform a Polymerase Chain Reaction (PCR). This technique works by cycling a mixture of DNA Template, Primers, Taq Polymerase, Magnesium Chloride, and dNTP's through three specific temperatures to create more copies of the desired sequence. After assembling the PCR mixture, the PCR machine was programmed to perform three stages. In the first stage, the samples went through one cycle at 95⁰C for 3 minutes. The purpose of this stage is to warm up the machine and prepare the samples for denaturing. The second stage puts the samples through 35 cycles of 95⁰C for 30 Seconds, 57⁰C for 30 seconds, and 72⁰C for 30 seconds. The purpose of the first part of the second stage is to break apart the hydrogen bonds between the base pairs, denaturing the DNA sequence into two separate strands. The purpose of the low temperature is to allow primers to bind. The purpose of the middle temperature is to create an environment for Taq Polymerase to assemble a new strand. Stage three puts the samples through one cycle of 72⁰C for 3 minutes. There is a final hold of 4⁰C. Take the samples out of the PCR machine. The target sequence has been amplified a million times! To analyze the sequence, see the Fluorimeter section.


Fluorimeter Measurements

To analyze the DNA samples after they've been amplified, A smartphone with adjustable settings was used for this procedure.
Using the provided smartphone 'cradle', place the smartphone in it and set the phone to these settings:

- Set exposure to highest setting
- Set ISO to 800+
- Set white balance to Automatic
- Set saturation to highest setting
- Turn the flash off if the camera is equipped
- Set contrast to lowest setting


The Fluorimeter aparatus consists of a hydrophobic Teflon surface on a glass slide. There is an array of 3x10 glass circles on the surface to anchor the drops. First, place two drops of the cybergreen solution on row 2, column 1&2 (Figure 1). With a sterile pipet, place two drops of amplified sample DNA on top of the cybergreen solution. Do not cross contaminate DNA samples! Take a picture using the settings listed above. Prepare for another sample by sucking the liquid off the slide using a pipet and moving the slide back by two columns. Continue down the columns with the cybergreen drops, another DNA sample(with a new, sterile pipet), pictures, and preparations for another sample. This procedure will require using two slides.


Interpreting results:

If the dot has DNA, the dye with LED will make the drop appear green. Water will glow blue, and a negative result will also not be green

Research and Development

Specific Cancer Marker Detection - The Underlying Technology

Polymerase Chain Reaction, or PCR, is used to ampllify a specific segment of DNA, in this case the segment known to code for cancer. A primer, which is an artifically synthesized piece of complementary DNA, binds to the desired DNA segment, and the enzyme taq polymerase catalyzes the replication of the DNA strand using dNTPs (free bases). This is repeated over and over to produce many copies of the DNA. Once the copies are made, a fluorescent dye that only bonds to DNA double strands is added to the solution. If a solution that shows fluorescence by using a fluorimeter, then that DNA sample contains the cancer-associated sequence.

The single nucleotide polymorphism, or SNP, that is linked to cancer is rs17879961. The DNA base sequence associated with cancer is ACT, while the non-cancer sequence is ATT. PCR works to identify ACT from ATT because the primers, which are complementary to DNA strands containing cancer-positive ACT, will not bind to DNA containing ATT, and instead of DNA double strands, the solution will only contain single strands. The fluorescent dye will only bind to double strands and will identify those.



Adapted from: http://users.ugent.be/~avierstr/principles/pcr.html



Results

Image:fluorimeterexcel.jpg


These Images were taken using a HTC evo4G using the settings described under flourimeter measurements.

Water and SYBR green

Image:fluorimeterarray.jpg

Water and SYBR green under split color conditions for green Image:WATERSYBR.jpg

SYBR green and Calf Thymus

Image:CalfThumus.jpg


Sample Integrated Density DNA μg/mL Conclusion
PCR: Negative Control E6 F6 G6
PCR: Positive Control E7 F7 G7
PCR: Patient 1 ID #####, rep 1 E8 F8 G8
PCR: Patient 1 ID #####, rep 2 E9 F9 G9
PCR: Patient 1 ID #####, rep 3 E10 F10 G10
PCR: Patient 2 ID #####, rep 1 E11 F11 G11
PCR: Patient 2 ID #####, rep 2 E12 F12 G12
PCR: Patient 2 ID #####, rep 3 E13 F13 G13


KEY

  • Sample =
  • Integrated Density = Using the imageJ software, the image was split into its 'red', 'green' and 'blue' color components. This value represents the amount of 'green' light measured from the excitation through the blue LED light shining through the sample and subtracted from the background value, which was 'black'
  • DNA μg/mL =
  • Conclusion =
Personal tools