BME100 s2014:W Group12 L5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: RJ Parkinson
Name: Jacqueline Stokes
Name: Austin Doyle
Name: Anthony Zlatket
Name: Sara Jerez


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
The SYBER I Green dye is used in this lab to visually detect amplified samples of DNA. SYBER green acts as a dye molecule that emits a florescent green color in the presence of double stranded DNA. To detect this light the sample is mixed with SYBER green and placed under a black box to block out interfering surrounding light. Then using a smartphone to take an image of the mixture the we can analyze the brightness of the dye and how much of the dye fluoresces to indicate the amount of DNA in the sample.


Single-Drop Fluorimeter
This lab uses a single drop fluoremeter device to detect DNA samples. This device consists of a sturdy outer cover which serves to block out interfering light sources so that the fluorescent light is more easily detected. We set up a phone cradle to secure the phone while it is taking pictures. Inside the device there is a platform for the slides to be placed on a surface designed to repel water so droplets on the slide maintain a spherical shape. Around the slide there is a LED light source which emits a blue light in order to clearly see the fluorescing dye.

How the Fluorescence Technique Works
The blue LED light shines on the sample underneath the black box, in the dark, and detects the different DNA concentrations. The samples are shown to have different DNA concentrations based on the amount of light that they fluoresce.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5C
    • Flash: None
    • ISO setting: Auto
    • White Balance: Auto
    • Exposure: Auto
    • Saturation: Auto
    • Contrast: Auto
  • Did not arrange the settings on the phone. But the lab manual says that this is acceptable because the camera is most likely sensitive enough.



Calibration

The phone is placed upright in the cradle with an eraser to help it stand straight. The cradle is moved about 6.5 cm away from the fluorimeter. This set up never moves once the experiment has begun.

  • Distance between the smart phone cradle and drop = 6.5 cm


Solutions Used for Calibration

row 1 cell 1 row 1 cell 2 row 1 cell 3 row 1 cell 4
row 2 cell 1 row 2 cell 2 row 2 cell 3 row 2 cell 4
row 3 cell 1 row 3 cell 2 row 3 cell 3 row 3 cell 4

Add more rows as needed


Placing Samples onto the Fluorimeter

 Step 1: Insert the slide, smooth side down
 Step 2: 80 milliliters of the SYBR GREEN on the first slide position
 Step 3: 80 milliliters of the calf thymus (water blank) on top of the "beach ball" of SYBR GREEN
 Step 4: Focus camera on the "beach ball", cover the fluorimeter with the black box, and take a picture in the dark
 Step 5: Repeat this process on different slide placements (at least three) for each sample.


Data Analysis

Representative Images of Samples

Image with No DNAImageJ with NO DNA.PNG

Image with DNAImageJ with DNA.PNG



Image J Values for All Samples

PCR Product Tube Label Diluted PCR Product solution volume (µL) SYBR Green 1 Dye solution volume (µL) INTDENS values INTDENS values INTDENS values Average INTDENS value
RJ1 80 80 925556 370350 NA 647953
RJ2 80 80 80765 52661 30813 54746
RJ3 80 80 47062 39069 51594 45908
RJ4 80 80 54742 31713 58904 48786
RJ5 80 80 50946 56850 51390 53062
RJ6 80 80 58585 57683 65830 60699
RJ7 80 80 54772 55469 97643 59294
RJ8 80 80 56383 54353 57477 56071


Fitting a Straight Line

BME100 12 Calibration.jpg


PCR Results Summary

Our positive control PCR result was 30.20 μg/mL. Our negative control PCR result was -36.08 μg/mL

Patient 78086 : The images of the three samples of DNA given from Patient 78086 were without color. There was no sign of any SYBR Green 1 in all three images. The amount of corrected PCR product concentration was around -36 μg/mL. The exact values were: -35.89, -36.88 and -36.56 μg/mL.

Patient 32445 : The images of the three samples of DNA given from Patient 32445 were without color as well. Again there was no sign of any SYBR Green 1 in all three images. The amount of corrected PCR product concentration was around -35 μg/mL. The exact values were: -35.23, -35.38 and -35.74 μg/mL.

Patient 78086 : Negative. We were able to come to this conclusion based on the values of μg/mL for both the positive and negative controls. This patient's corrected PCR product concentration matched that of the negative control.

Patient 32445 : Negative. We were able to come to this conclusion based on the values of μg/mL for both the positive and negative controls. This patient's corrected PCR product concentration matched that of the negative control as well. We did not get confused on the results and assume that the results of the patients would be different. They in fact were very similar and we were able to successfully ascertain the desired result.