BME100 s2014:W Group7 L5

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png BME 100 Spring 2014 Home
People
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
Photos
Wiki Editing Help
BME494 Asu logo.png


OUR TEAM

Name: Haley Sivertson
Name: Velia Butruz
Name: Matthew Welz
Name: Valerie McDonald
Name: Elizabeth Hansen


LAB 5 WRITE-UP

Background Information

SYBR Green Dye

SYBR Green Dye was the indicator used in this experiment. As it binds to DNA, it emits green light clearly when introduced to double-stranded DNA. However, when in the presence of single strands of DNA, RNA, or water, SYBR Green I glows significantly weaker or not at all.


Single-Drop Fluorimeter

A single-drop fluorimeter was the device used in this experiment. It measures the emission of light; in this experiment, it was used to determine the emission of green light from SYBR Green I. The value of the emission is related to the amount of the florescent or the amount of material being identified. If the amount of florescent is identical between samples, then the value of the light emission between samples shows the proportionality of the material being identified. In this pursuit, the amount of SYBR Green I was identical between samples, and the single-drop fluorimeter was utilized to determine the presence of double-stranded DNA (as SYBR Green I glows brightly in double-stranded DNA as compared to other substances).


How the Fluorescence Technique Works

Spectrofluorometer is extremely flexible, providing continuous ranges of excitation and emission wavelength. The light from an excitation source passes through a filter or monochromatic, and strikes the sample. A proportion of the incident light is absorbed by the sample, and some of the molecules in the sample fluoresce. The fluorescent light is released in all directions.


Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Apple iPhone 5S
    • Flash: OFF
    • ISO setting: Auto
    • White Balance: Auto
    • Exposure: Auto
    • Saturation: Auto
    • Contrast: Auto


Calibration

The iPhone was placed in the cradle for support, allowing the camera to point directly at the slide at a right angle. The height of the fluorimeter was adjusted accordingly to allow for the best angle for the test.

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


Solutions Used for Calibration

Concentration.png


Placing Samples onto the Fluorimeter

  1. Step one consisted of setting up the camera in the cradle and aligning it at a certain length away from the light that shines on the drops of liquid on the slide.
  2. The second step was making sure all the samples were properly mixed into the different solutions.
  3. The third step was to place 80 micro liters on the slide with the rough hydrophobic side right in the middle of the beam or light.
  4. Step four was making sure to cover the sample and that no light was coming in and reaching the solution so the camera could take the picture of the sample. Steps 1-4 were repeated until all solutions were completed.


Data Analysis

Representative Images of Samples

Sample with no DNA


---screenshot007.png


Sample with DNA


+++screenshot007.png


Image J Values for All Samples


Original Calibration

Final DNA Concentration RAWINTDEN of the Drop RAWINTDEN of the Background RAWINTDEN (drop) - RAWINTDEN (background)
0 345313 30224 315089
0 180564 6007 174557
0.25 491145 21842 469303
0.25 397249 24163 373086
0.5 422049 21030 401019
0.5 508450 20911 487539
1 600240 28758 571482
1 591941 31183 560758
2 630004 34042 595962
2 581118 28959 552159
5 667090 46900 620190
5 711795 34870 676925


Quick Calibration

Final DNA Concentration RAWINTDEN of the Drop
5 959866
5 889122
2 408293
2 829977
1 356280
1 658943


PCR Product Sample Measurements

PCR Product TUBE LABEL Volume of the DILUTED PCR Product solution (µL) Volume of the SYBR GREEN I Dye solution (µL) INTDENS VALUES BASED ON 3 SEPARATE DROP MEASUREMENTS ' ' AVERAGE INTDENS VALUE
1A 80 80 256371 412299 165141 154247
2A 80 80 388143 377660 332221 366008
3A 80 80 238397 188553 437579 288176.333
1B 80 80 219121 484088 354011 352406.6667
2B 80 80 369201 331460 355380 352013.6667
3B 80 80 378003 335356 349226 354195



Fitting a Straight Line

This graph represents the relationship between concentration and RAWINTDEN.


Concentration vs RAWINTDEN graph.png


    1. Your positive control PCR result was 0.2306 μg/mL
    2. Your negative control PCR result was -0.066416 μg/mL


Write-in each patient ID and give both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed

    1. Patient 32298 : This patient did not produce a fluorescent light when the green dye solution was added to the slides. There was an average PCR product concentration of -0.11065 μg/mL.
    2. Patient 26685 : This patient also did not produce a fluorescent light when the green dye solution was added to the slides. There was an average PCR product concentration of -0.04375 μg/mL.

Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion.

    1. Patient 32298 : After comparing the two controls, it can be concluded that Patient 32298 does not contain the DNA strand because the patient's PCR Concentration values were negative and close to that of the negative control value.
    2. Patient 26685 : After comparing the two controls, it can be concluded that Patient 26685 also does not contain the DNA strand because this patient's PCR Concentration values were also negative and close to that of the negative control value.