BME100 f2013:W900 Group6 L5

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OUR TEAM

Name: James Mendoza
Name: Brandon Kehring
Name: Courtney DuBois
Name: Jacob Hulett
Name: Areli Isherwood


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR green dye is used in molecular biology as a nucleic acid stain. The dye generally binds to double-stranded DNA, while staining single-stranded DNA. The DNA-dye-complex that forms emits a green light (wavelength of approx. 520 nm) and absorbs blue light (wavelength of approx. 497 nm). SYBR green dye is commonly used in various methods of quantitative PCR.

Single-Drop Fluorimeter
A device that measures the fluoresence within a given drop of DNA solution. The quantity of fluorescence is generally directly related to the amount of florescent material in the given solution. The fluorimeter is capable of measuring the intensity and wavelength distribution of the spectrum that is emitted when the light passes through the DNA solution.

BME100 group6 device.jpeg

How the Fluorescence Technique Works
The fluorescence technique works by using a dye that makes the proteins glow a fluorescent green, showing whether the protein is either present or is not.



Procedure

Smart Phone Camera Settings

  • Model: HTC One
  • Flash: No
  • ISO setting: 100
  • White Balance: Fluorescent
  • Exposure: 0 (Default)
  • Saturation: 0 (Default)
  • Contrast: 0 (Default)

Calibration

We set the light and slides about 7 cm from the smartphone camera and we set the box over it to block out the light.

  • Distance between the smart phone cradle and drop = 7cm

BME100 Group6 Fluorimeter.jpg

Solutions Used for Calibration

Calf Thmus Dna solution concentration (microg/mL) Volume of the 2X DNA solution (microliters) Volume of the SYBR GREEN I Dye solution (microliters) Final DNA concentration in SYBR GREEN I Assay (ng/mL)
5 80 80 2.5
2 80 80 1
1 80 80 0.5
0.5 80 80 0.25
0.25 80 80 0.125
0.125 80 80 blank


Placing Samples onto the Fluorimeter

  1. Place the slide in its designated place, smooth side down, into the single droplet Fluorometer device.
  2. Micro-pipette 80µL of the SYBR green dye and place it in the second row of the middle column of the slide.
  3. Next carefully Micro-pipette 80µL of desired concentration of calf thymus DNA on top of the SYBER green dye onto the slide.
  4. The total amount of SYBR green dye/ calf thymus DNA mixture will be 160µL
  5. Start test by taking a photo of the droplet.


Data Analysis

Representative Images of Samples
No DNA BME100 Group6 WithoutDNA.png

With DNA BME100 Group6 WithDNA.png

Image J Values for All Samples

Calf Thymus DNA Concentration (FINAL), μg/mL Image Number AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND
2.5 1 33691 132.209 4454265 4454265
2.5 2 33840 131.203 4439919 4439919
1 1 36244 79.188 2870091 2870091
1 2 43552 79.813 3476030 3476030
0.5 1 37294 37.722 1406812 1406812
0.5 2 31940 38.819 1239877 1239877
0.25 1 43038 23.1 994189 994189
0.25 2 35870 27.141 973557 973557
0.125 1 41800 6.658 278307 278307
0.125 2 39159 5.107 199977 199977
0 1 44228 6.221 275159 275159
0 2 39651 7.252 287535 287535

Note: Only two images needed per Dr. Garcia since there were initial problems with the HD camera


Fitting a Straight Line BME100 Group6 CalibrationGraph.jpg