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
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
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
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.
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.
Step three: Attach a new tip to the micropipette and draw 80 microliters of Calf Thymus solution into the micropipette.
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
Figure 1:Photograph of 0 (ng/mL) concentration DNA sample highlighted by the area selection tool in ImageJ.
Figure 2:Photograph of 2.5 (ng/mL) concentration DNA sample highlighted by the area selection tool in ImageJ.
BME 100 Fall 2013
