Name: Nicole Phillips Role: Leader, Experimental Protocol Professional
Name: Francis Taguinod Role:Research and Development Adept
Name: Christian "Chin-Chin" Kaiser Role: PCR Machine Specialist
Name: Augusta Dumanski Role: Team Mascot, Experimental Technician
Name: Christiana Montoi-Reeves Role: Research and Development Guru
LAB 5 WRITE-UP
Background Information
SYBR Green Dye The SYBR green dye is a dye which glows green when exposed to DNA, specifically the nucleic acids in DNA. The more DNA present, the brighter the glow.
Single-Drop Fluorimeter The Single-Drop Fluorimeter is a small box with a slot for a multi-welled slide to fit in at the top. There is a blue LED light which shines across where the slide fits in which can be turned on by a switch on the right hand side of the box. The purpose of this flourimeter is to illuminate drops placed on the slide by aligning them properly with the light.
How the Fluorescence Technique Works The DNA and dye are placed on the slide using a micropipetter in the center of two dots in the middle of the slide. The rough texture of the slide and the holes placed throughout allow the liquid to hold its shape. The slide is then positioned so that the light shines through the drop and a picture is taken in the dark of the drop to get a reading on how much DNA is present based on how bright the sample glows.
Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone 5
Flash: Off
ISO setting: normal
White Balance: normal
Exposure: 200%
Saturation: normal
Contrast: normal
Calibration
Place the camera a set distance in front of the flourimeter sideways, use the placeholder for the camera to stay in place. Place the box on top of the flourimeter and the phone, set a timer for three seconds and close all flaps. Wait for the camera to snap a picture and remove the box and repeat the steps.
Distance between the smart phone cradle and drop = 15 cm
Solutions Used for Calibration[Instructions: See worksheet page 6.]
Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL)
Volume of the 2X DNA solution (µL)
Volume of the SYBR GREEN I
Final DNA concentration in SYBR Green I solution (µg/mL)
Dye solution (µL)
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
0
[Add more rows as needed]
Placing Samples onto the Fluorimeter
Set a slide on the fluorimeter for the blue light to be in between two and three holes
Apply a tip on the micropipette
Insert the sample onto the tip
Eject the sample on the slide
Remove the tip and dispose in the proper waste disposal
Take a sample of SYBR Green I on a new tip
Eject the SYBR Green I on the slide with the sample
Properly dispose of the tip after emptying the SYBR Green I
BME 100 Spring 2014
