BME100 f2013:W1200 Group9 L5

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OUR TEAM

Name: Jessica Stradford
Name: Kenna Lum
Name: Rachael Hall
Name: Michelle Sigona
Name: Salvador Avina


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR Green Dye is a cyanine dye used in many molecular biology and biochemistry experiments as a nucleic acid stain. The dye is able to detect polymerase chain reactions products by binding to DNA by absorbing the blue light and emitting the green light, binding to DNA that is double stranded but still having the capability to stain single-stranded DNA and RNA. The PCR products are created and during the PCR the DNA polymerase amplifies the target sequence where the dye then binds to every new copy of double-stranded DNA. As the process continues along, more PCR product is created. One of them main disadvantages to the dye is the fact that the dye binds to strands that are nonspecific double-stranded DNA, producing what is known as a false positive meaning that you need to ensure the product is a well-designed primer for the best results with the DNA. Another problem we saw with the dye was that the color seemed to have "washed out" from the lighting in the room we were working with so we did not see any results until we switched to a different dye.


Single-Drop Fluorimeter
A Single-Drop Fluorimeter device is a black box with a small slit at the top where a glass slide can fit into it. A sample can be placed on this glass slide where a blue LED light will shine onto the sample. The device then has a big black box to keep out the light from entering from the slides, and then a photo can be taken of the sample. The fluorimeter works by using detectors to measure the wavelengths of fluorescence through the use of light, measuring the light absorbed and the light emitted. IMG 0116.jpg


How the Fluorescence Technique Works
When beginning the experiment, make sure the hydrophobic side of the slide is face up and place a drop of the dye on that side of the slide in the fluorimeter. The dye clumps together, forming one droplet on the slide and after adding the DNA to the droplet, the dye should exhibit a florescent appearance. The fluorescence is visible because the dye is placed on the slide where outside lighting is eliminated and instead, uses the light beams from the slide. A short wavelength is emitted from this fluorescent, producing photons. From these photons, the energy released can determine the samples' concentrations. The results can then be analyzed by the fluorescence in the pictures taken and we will then be able to identify the directly proportional relationship of the DNA and fluorescence.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Galaxy S4
    • Flash: off
    • ISO setting: 800
    • White Balance: Auto
    • Exposure:+2.00
    • Saturation:N/A
    • Contrast:N/A


Calibration
In order to calibrate the phone to the fluorimeter, we used the guess and check method. Due to a lack of plastic plates to raise the fluorimeter, we used a box that at one point contained tips for the micropipette. We then elevated it further with a plastic plate. The phone cradle was also raised by a plastic plate, in order for us to get the right angle for the side profile shot of the drop. Therefore, the final set up was the fluorimeter on a plastic box stacked with a plastic plate, and the phone cradle slightly elevated by a plastic plate.


Setup 9PM.jpg

  • Distance between the smart phone cradle and drop = 12 cm


Solutions Used for Calibration

Calf Thymus DNA Solution Concentration (microg/mL) Volume of the DNA Solution (uL) Volume of the SYBR Green I Dye Solution (uL) Final DNA Concentration in SYBR Green I Assay (ng/mL)
5 80 80 2.5
2 80 80 1
1 80 80 0.5
.5 80 80 0.25
.25 80 80 0.125
0 80 80 blank


Placing Samples onto the Fluorimeter

  1. Place the slide smooth side down in the fluorimeter
  2. Adjust the device so that the camera, when placed in the cradle, would be able to take a photo of the drop from a side view
  3. Turn on the light using the switch on the side of the device
  4. Put 80 uL of SYBR Green I solution, placed in between the two first dots in the middle column
  5. Add 80 uL of the sample/calibration solution to the 80 uL of SYBR Green I, so that there is a drop with a total volume of 160 uL
  6. Center the light in the middle of the drop and place the cradle 12 cm away from the center of the drop
  7. Take 3 pictures per drop and repeat the steps again for another sample of the same concentration to total 2 trials with 3 pictures each.
  8. Repeat steps 4-8 for each of the 6 concentrations: 0, .25, .5, 1, 2, and 5.


Data Analysis

Representative Images of Samples

Zero DNA
Group90.jpg

Positive with DNA
Group91.jpg


Image J Values for All Samples

' Area Mean IntDen RawIntDen RawIntDen (background)
0.25 11384 46.578 530249 530249 5175
0.25 11440 64.73 740508 740508 11446
0.25 11232 50.704 569503 569503 2323
0.5 9464 41.894 396489 396489 4157
0.5 9596 41.748 400612 400612 4843
0.5 10172 100.752 1024845 1024845 2042
0 11240 29.108 327174 327174 5092
0 11552 30.266 349631 349631 4618
0 10932 68.733 751394 751394 6072
1 7912 26.805 212084 212084 3680
1 10868 27.285 296535 296535 6739
1 8188 26.943 220613 220613 5709
2 10260 33.225 340884 340884 4932
2 10564 35.492 374942 374942 5164
2 9260 32.379 299828 299828 3116
5 11152 72.496 808471 808471 6219
5 11552 72.826 841282 841282 5407
5 10056 78.562 790018 790018 3258


Fitting a Straight Line

Dnacalibrationgroup9.jpg