BME103:T130 Group 11

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(OUR TEAM)
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| [[Image:BME103student.jpg|100px|thumb|Name: Timothy Peterson<br>R and D]]
| [[Image:BME103student.jpg|100px|thumb|Name: Timothy Peterson<br>R and D]]
| [[Image:BME103student.jpg|100px|thumb|Name: Sharon Gooi<br>Role: Experimental Protocol Planner]]
| [[Image:BME103student.jpg|100px|thumb|Name: Sharon Gooi<br>Role: Experimental Protocol Planner]]
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| [[Image:BME103student.jpg|100px|thumb|Name: Derek Biah<br>Role: Data Analyzer]]
| [[Image:BME103student.jpg|100px|thumb|Name: student<br>Role(s)]]
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Revision as of 13:48, 13 November 2012

BME 103 Fall 2012 Home
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
Course Logistics For Instructors
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Contents

OUR TEAM

Name: Timothy PetersonR and D
Name: Timothy Peterson
R and D
Name: Sharon GooiRole: Experimental Protocol Planner
Name: Sharon Gooi
Role: Experimental Protocol Planner
Name: Derek BiahRole: Data Analyzer
Name: Derek Biah
Role: Data Analyzer
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
Role(s)

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:PCR.png

The PCR machine heats up the DNA so that enzymes can "unzip" the two strands of DNA. This process happens in cycles so that the DNA will seperate and duplicate a multitude of times. A certain amount of primer is used to duplicate the DNA specific to the amount of original DNA. By amplifying the amount of DNA, a proper diagnosis of a certain gene can be made.


Experimenting With the Connections

When we unplugged the LCD screen from the circuit board, the machine's screen shut off.

When we unplugged the white wire that connects the circuit board to tube PCR block, the machine stopped reading the temperature.

Test Run

We first tested the PCR machine on the 18th of October, 2012. The LCD screen readings matched the reading on the computer PCR program, and the machine worked well and efficiently.




Protocols

Polymerase Chain Reaction (PCR)


The Polymerase Chain Reaciton (PCR) is a process that depends on a DNA Polymerase enzyme's ability to synthesize a strand that is complementary to a targeted fragment of DNA in a test tube mixture of all four DNA bases, which are adenine, cytosine, guanine and thymine. Besides that, the test tube mixture also must have two fragments of DNA of about 20 base pairs that are called primers. These primers should have sequences complementary to adjacent areas of each side of the targeted DNA segment. Since these two primers should match exactly with only the targeted DNA sequence, only this area would be defined and copied.


For the process of PCR, there are different stages, and they are collectively called the heating-cooling cycle. First of all, the mixture would be heated to separate the sides of the double-stranded DNA. After that, the mixture would be cooled to an optimum temperature for the primers to find and bind to whichever side of the separated DNA strands that they are complementary to. The temperature is then raised slightly to reach an optimum temperature for the polymerase, which is included in the test tube mixture, to extend the primers so that new complementary strands are generated. At the end of these first cycle, there will be two copies of the targeted DNA segment. The cycle is repeated multiple times to generate more and more copies of the targeted DNA segments. With the advent of technology in biological sciences and engineering, the entire process can be automated after all the correct components are added into a tube by using a thermocycler or a PCR machine such as the OpenPCR machine.



Reagent Volume
Template DNA (20ng) 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


During Week 2, there were eight samples that PCR was run on. These samples consisted of a positive control, a negative control, three samples from Patient 1 and three samples from Patient 2.


Patient 1

ID #: 11640

Gender: Female

Age: 54 years old


Patient 2

ID #: 29292

Gender: Male

Age: 63 years old



Fluorimeter


The eight samples from the Polymerase Chain Reaction experiment were used in this experiment. In addition to that, eight Eppendrof tubes filled with 400ml of buffer to maximise fluorescence, a Eppendorf tube filled with DNA (calf thymus standard at 2 micrograms/ml; the tube was marked with a red dot on the cover), water in a scintillation vial, an Eppendorf tube filled with SYBR GREEN 1 (marked with a blue dot on the cover), several glass slides, a fluorimeter, a black box, a smartphone stand, a smartphone, a marker pen and several pipettes, a pipette with a blue strip, a pipette with a red strip, a pipette with a black strip and a pipette with a blank piece of paper taped to it, as well as a cup were used.


The eight Eppendrof tubes were labeled using the marker pen according to the eight samples from the Polymerase Chain Reaction experiment; they were labeled +, -, 1a, 1b, 1c, 2a, 2b and 2c. Similarly, for each Eppendorf tube (ten altogether), a pipette for each tube was given a corresponding label. Using the corresponding pipettes, the eight samples from the Polymerase Chain Reaction experiment were transferred from their PCR tubes to their corresponding Eppendrof tubes. The pipettes were all kept carefully separate from each other.


The fluorimeter was set up according to the image shown.


A complete setup of the Fluorimeter


The pipette with the blue strip was used to put two large drops of SYBR GREEN I on the first two centered drops on the glass slide in the fluorimeter.(Warning: please do not dispose of any of the pipettes used until the entire process is complete!) Once that was done, The corresponding pipette for the positive control was used to add two drops of the sample of the positive control to the two drops of the SYBR GREEN I that was already on the glass slide. The light in the fluorimeter was aligned to ensure that it was going through the drop. The fluorimeter was covered with the black file box and the smartphone operator was allowed to take as many pictures as needed. After the pictures were taken, the pipette with the black strip was used to dispose of the drop on the glass slide into the cup. The slide was then pushed forward so that the light would be in the general direction of the next two centered holes on the glass slide.


The above process was then repeated using the next samples available, which were the negative control sample, sample 1a, 1b, 1c, 2a, 2b and 2c. Once the first five samples were done, shifting the glass slide down two holes after every sample, that glass slide was disposed off, and a new glass slide was used.


After these eight samples were run, two drops of SYBR GREEN I were put on the appropriate two centered drops before two drops of the Calf Thymus DNA from the Eppendrof tube with the red dot on top were added using the pipette with the red strip, and the above procedure was repeated. Lastly, after the slide was moved two holes down and two drops of SYBR GREEN I was added to the slide, the pipette with a blank piece of paper taped to it was used to add two drops of water from the scintillation vial on the slides, with the above procedure also being repeated on it. After all of this was done, the slides, pipettes, and all of the tubes containing the samples were disposed of in the correct waste containers.

Flourimeter Measurements

Tubes


Description Eppendorf Tube Label Pipette Label
Positive Control + +
Negative Control - -
Patient 1 Sample 1 1a 1a
Patient 1 Sample 2 1b 1b
Patient 1 Sample 3 1c 1c
Patient 2 Sample 1 2a 2a
Patient 2 Sample 2 2b 2b
Patient 2 Sample 3 2c 2c


DNA Measurement Operator: Smartphone


Image Number 2 Drops SYBRGr 2 Drops Comments
1A Patient 1 Sample 1 Darker blue tone with dark vertical streaks
1B Patient 1 Sample 2 Lighter Blue tone with very thin, vertical, fluorescent streaks
1C Patient 1 Sample 3 Lighter Blue tone with very fine, hazy fluorescent streaks
2A Patient 2 Sample 1 Light blue tone with thick,vertical, hazy, fluorescent streaks
2B Patient 2 Sample 2 Darker blue tone of drop with a variation of thick and fine, vertical, fluorescent streaks
2C Patient 2 Sample 3 Darker toned drop with very fine, wispy, fluorescent streaks
Positive Control (+) Positive Control Lighter blue drop with green round shape blobs displaced throughout
Negative Control (-) Negative control Lighter Blue drop with light, vertical streaks. No apparent fluorescence.
Water (H2O) Water Dark blue drop with no fluorescent streaks.
Calf Thymus Calf Thymus Dark blue drop with hazy, green, abstract round shapes distributed around the blob.


ImageJ Software Processor


Sample or Background ID Area and x, y, w, h, info Mean Pixel Value INTDEN RAWINTDEN INTDEN (IF DIFFERENT)
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Data Analyzer


Description INTDEN with background subtracted DNA Concentration, micrograms/ml
WATER BLANK 0
DNA CALF THYMUS, 2 microg/ml 2
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Research and Development

Specific Cancer Marker Detection - The Underlying Technology

PCR helps to detected certain types of genes. In this case it is used to find out a specific type of cancer. In the process of detecting the cancer, primers are made to compliment a DNA strand that has the cancer gene in it. If a subject has the cancer in their DNA, the primers will bind to strand, whereas a subject without the cancer would not have a primer attach to their DNA strand.

The r1787996 SNP is linked to the cancer sequence. The codon ATC is the sequence for cancer where the ATT means there is no cancer. In PCR, the ATC cancer sequence is detected because the primers will only attach to the DNA strands that have the ATC sequence. The ATT, non-cancer, strands will not bind with the primers. Only the combined primer DNA strand will be detected thus alerting for cancer.

(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.)




Results

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 =
  • DNA μg/mL =
  • Conclusion =


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