BME100 f2013:W900 Group6 L5

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Contents

OUR TEAM

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

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

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

With DNA

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
2.5133691132.20944542654454265
2.5233840131.20344399194439919
113624479.18828700912870091
124355279.81334760303476030
0.513729437.72214068121406812
0.523194038.81912398771239877
0.2514303823.1994189994189
0.2523587027.141973557973557
0.1251418006.658278307278307
0.1252391595.107199977199977
01442286.221275159275159
02396517.252287535287535

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


Fitting a Straight Line

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