BME100 f2013:W900 Group8 L5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: Kendall Hickie
Roles: Procedure, Photographer
Name: Ashley Ivany
Roles: Prodedure, Pipette Specialist
Name: Samuel Butler
Roles: Background Information, Setup
Name: Connor Mccoy
Roles: ImageJ 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
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

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

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

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
2.5 image 1 22986 111.42 2485056 22007 2463049
2.5 image 2 24100 109.472 2710241 25678 2684563
2.5 image 3 25732 104.396 2442879 32211 2410668
1 image 1 21794 95.555 1918914 24669 1894245
1 image 2 24264 87.353 1931095 26286 1904809
1 image 3 22732 91.288 2099121 25697 2073424
0.5 image 1 21006 55.9 1126828 21621 1105207
0.5 image 2 19464 55.785 1130472 20187 1110285
0.5 image 3 23428 53.032 1110818 21018 1089800
0.25 image 1 22096 28.57 562225 21164 541061
0.25 image 2 19314 29.731 529745 11591 518154
0.25 image 3 17200 32.741 581305 23614 557691
0.125 image 1 20394 8.348 158373 23178 135195
0.125 image 2 20792 8.527 176981 23021 153960
0.125 image 3 19314 8.609 144964 23303 121661
0 image 1 19093 3.115 58337 13683 44654
0 image 2 19615 3.975 76550 30550 46000
0 image 3 17554 2.406 57211 11768 45443


Fitting a Straight Line
Group 8 Standard Curve.png