BME100 f2013:W900 Group8 L5

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Contents

OUR TEAM

Name: Kendall Hickie  Roles: Procedure, Photographer
Name: Kendall Hickie
Roles: Procedure, Photographer
Name: Ashley Ivany  Roles: Prodedure, Pipette Specialist
Name: Ashley Ivany
Roles: Prodedure, Pipette Specialist
Name: Samuel Butler  Roles: Background Information, Setup
Name: Samuel Butler
Roles: Background Information, Setup
Name: Connor Mccoy  Roles: ImageJ Specialist, Data Analysis
Name: Connor Mccoy
Roles: ImageJ Specialist, Data Analysis
Name: Kavin M Watson  Roles: Assistant Pipette Specialist, Data Analysis
Name: Kavin M Watson
Roles: Assistant Pipette Specialist, Data Analysis


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR Green Dye is a asymmetrical cyanine dye which is used as a nucleic acid stain. This provides a visible outline of the calf Thymus DNA solution.

Single-Drop Fluorimeter
Image:8pic3.jpg
The Single-Drop Fluorimeter is composed of a small black box, with an apparatus above which allows for multi-celled wall slide to fit. The switch on the side of the box allows for the blue LED light to be turned on and off. This LED light can be aligned accordingly, allowing for the LED light to shine through the DNA solution placed above.


How the Fluorescence Technique Works
The Fluorometer consists of a slide with holes where the DNA solution is placed. The DNA solution is able to maintain its round shape due to the Teflon layer on the slide. This layer allows the drop to maintain its original size (much like a drop). This allows for the LED light to shine through the DNA solution allowing it to glow. This then makes it much easier to observe and collect data as photos are taken of each trial.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Galaxy S3
    • Flash: Off
    • ISO setting: 800 (Highest Setting)
    • White Balance: Auto
    • Exposure: +2.0 (Highest setting)
    • Saturation: Unable to change
    • Contrast: Auto Contrast Off


An additional phone was not used due to the lack of adequate camera technology.


Calibration

Image:BME_100_Lab_Setup.jpg

  • Distance between the smart phone cradle and drop = 8.2 cm


Solutions Used for Calibration

Calf Thymus DNA Solution Concentration (microg/mL) Volume of the 2X DNA Solution (μL) Volume of SYBR GREEN I Dye Solution (μL) Final DNA Concentration in SYBR GREEN I Assay (ng/mL)
0.0 80 80 Blank
0.25 80 80 0.125
0.50 80 80 0.25
1.0 80 80 0.50
2.0 80 80 1.0
5.0 80 80 2.5


Placing Samples onto the Fluorimeter

  1. Step one: Place the multi celled wall slide into the Single-Drop Flourimeter and position it so that the light passes through the first two rows on the slide.
  2. Step two: Draw 80 microliters of SYBR green solution into the micropipette and carefully release the solution onto the center of the first two row of the slide. Release the tip of the micropipette into a waste disposal container.
  3. Step three: Attach a new tip to the micropipette and draw 80 microliters of Calf Thymus solution into the micropipette.
  4. Step four: Release the Calf Thymus solution on top of the SYBR green drop that is already on the slide. Dispose of micropipette tip into the waste container.


Data Analysis

Representative Images of Samples

Image:0_DNA.jpg
Figure 1: Photograph of 0 (ng/mL) concentration DNA sample highlighted by the area selection tool in ImageJ.

Image:Lots_of_DNA.jpg
Figure 2: Photograph of 2.5 (ng/mL) concentration DNA sample highlighted by the area selection tool in ImageJ.

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 RAWINTDEN - BACKGROUND
2.5image 122986111.422485056220072463049
2.5image 224100109.4722710241256782684563
2.5image 325732104.3962442879322112410668
1image 12179495.5551918914246691894245
1image 22426487.3531931095262861904809
1image 32273291.2882099121256972073424
0.5image 12100655.91126828216211105207
0.5image 21946455.7851130472201871110285
0.5image 32342853.0321110818210181089800
0.25image 12209628.5756222521164541061
0.25image 21931429.73152974511591518154
0.25image 31720032.74158130523614557691
0.125image 1203948.34815837323178135195
0.125image 2207928.52717698123021153960
0.125image 3193148.60914496423303121661
0image 1190933.115583371368344654
0image 2196153.975765503055046000
0image 3175542.406572111176845443


Fitting a Straight Line
Image:Group_8_Standard_Curve.png




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