BME100 f2013:W1200 Group8 L5

From OpenWetWare

Jump to: navigation, search
BME 100 Fall 2013 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
Image:BME494_Asu_logo.png

Contents

OUR TEAM

Name: Colby Mark - Analysis Specialist
Name: Colby Mark - Analysis Specialist
Name: Andrew Wills - Experimental Specialist
Name: Andrew Wills - Experimental Specialist
Name: Madison Grayson - Data and Procedure Specialist
Name: Madison Grayson - Data and Procedure Specialist
Name: Hayden McIver - Experimental Specialist
Name: Hayden McIver - Experimental Specialist
Name: Isai Valdez - Research Specialist
Name: Isai Valdez - Research Specialist


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
Is a small molecular dye that fluoresces very well in the presence of dsDNA but fluoresces very weakly in water or with single strands of DNA. The dye binds to DNA which then absorbs blue light and emits green light.


Single-Drop Fluorimeter
Is an instrument that detects fluorescence, quantity is related to the amount of florescent material and, less directly, proportional to the amount of the molecule being detected.
Image:Flour.jpg

How the Fluorescence Technique Works
The Fluorescence technique invovles adding dye that attaches to the DNA, which when you put blue light through it, excites the molecules causing an emission of light. This then shows the concentration of DNA in the sample.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: HTC One
    • Flash: off
    • ISO setting: 800
    • White Balance:Auto
    • Exposure:High
    • Saturation:default
    • Contrast:default


Calibration

The camera of the HTC One phone was lined up level with the fluorimeter and the flash was turned off. A photo of this can be seen in the above introduction section. The phone was placed in a vertical position on the cradle. The test strip was elevated to match the height of the phone in the cradle.

  • Distance between the smart phone cradle and drop = The distance between the smart phone cradle and the drop had to be a minimum of 4 centimeters. Our distance was exactly 7 centimeters for every picture.


Solutions Used for Calibration

Calf Thymus DNA Solution Concentration (microg/mL) Volume of the 2x DNA Solution (µL) Volume of the SYBR GREEN I DYE Solution 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 80 80


Placing Samples onto the Fluorimeter
Step 1- Place the glass slide, smooth side down, onto the fluorimeter.
Step 2- Adjust the height of the phone and fluorimeter so that the picture will be of the side of the drop.
Step 3- Using the micro-pipette, place 80µL of the SYBR GREEN I on the slide, connecting the first two circles in the middle.
Step 4- Again using the micro-pipette, drop 80µL of the sample solution on top of the SYBR GREEN I drop.
Step 5- Position the slide on the fluorimeter, so that the light passes through the center of the drop and focuses on the other side
Step 6- Adjust the phone's distance and focus from the fluorimeter to allow for the best picture.
Step 7- Cover the fluorimeter with the lightbox, leaving one panel open for access.
Step 8- Press the shutter button on the phone and close the lightbox, using a timer on the phone to give you enough time to do so.
Step 9- Once the picture is taken, record which solution is in the photograph.
Step 10- Remove the drop from the slide and dispose of it appropriately.
Step 11- Move the slide to th next position and repeat the previous steps for each sample.


Data Analysis

Representative Images of Samples


Calf Thymus DNA solution concentration of 0 micro grams/mL


Image:With_no_DNA_2.jpg




Calf Thymus DNA solution concentration of 2 micro grams/mL


Image:With_some_DNA_2.jpg


Image J Values for All Samples


Calf Thymus DNA Concentration (FINAL), μg/mL ' AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND
0.25image 13335374.4472483023360236
0.25image 23335385.4512850044523085
0.25image 33273286.5622833363707536
0.5image 124263103.3472507512115797
0.5image 224263105.1682551685160892
0.5image 323734108.3022570430179709
1image 120188157.0733170995109397
1image 221164155.3793288445146047
1image 320967153.9033226875151989
2image 126476188.6834995584113277
2image 225922189.5614913801167964
2image 325796193.7914999027142127
5image 127488204.3595617421118420
5image 226257182.2574785510118784
5image 328646211.3196053453226367
0image 13836890.8233484683441468
0image 23425377.2172644917308348
0image 32558375.8111939470514997


Fitting a Straight Line

Image:Flourecence_data.jpg



Personal tools