Name: Madison Grayson - Data and Procedure Specialist
Name: Hayden McIver - Experimental 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.
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
Calf Thymus DNA solution concentration of 2 micro grams/mL
BME 100 Fall 2013
