SYBR Green Dye
SYBR Dye is a photosensitive molecular dye that exhibits a green fluorescence in the dark when added to enough single-stranded DNA.
Single-Drop Fluorimeter
A Single Drop Fluorimeter is a device that shines blue light through a drop sample that is placed on a glass slide and detects fluorescence.
How the Fluorescence Technique Works [Instructions: In your own words]
Procedure
Smart Phone Camera Settings [Instructions: The type of smart phone you used and how you adjusted the camera settings, if applicable (see worksheet page 4).]
Type of Smartphone: Samsung Galaxy S2
Flash: Off
ISO setting: Default
White Balance: Auto
Exposure: Highest
Saturation: Default
Contrast: Default
Calibration
The camera phone was set up in the cradle with an angle to view the side of the drop. The fluorimeter was raised about 1 1/4 inches to accomodate for the phone height.
Distance between the smart phone cradle and drop = 6.5 cm
The phone was removed from this image to simplify and completely show distance setup.
Solutions Used for Calibration
Distance of Phone from Fluorimeter
6.5 cm
Initial Concentration of 2X Calf Thymus DNA solution (μg/mL)
Volume of the 2X DNA solution (μL)
Volume of the SYBR Green I Dye Solution
Final DNA concentration in SYBR Green I Solution (μg/mL)
5.0
80
80
2.5
2.0
80
80
1.0
1.0
80
80
0.5
0.5
80
80
0.25
0.25
80
80
.0125
0.0
80
80
0.0
Placing Samples onto the Fluorimeter
[Instructions: Step one, in your OWN words]
[Instructions: Step two, in your own words]
[Instructions: Step three, in your own words]
[Instructions: Step etc., in your own words]
Data Analysis
Representative Images of Samples
High DNA - using green channel
Low DNA - using green channel
Image J Values for All Samples
[Instructions: See worksheet page 8. To save time on typing a new Wiki table from scratch, use THIS TOOL to auto-generate a Wiki table: Excel-to-Wiki Converter. Copy the headers and values from the Excel spreadsheet you made, paste them into the form field, click submit, copy the Wiki code that the tool generated, and replace TABLE GOES HERE (below) with your auto-generated code.]
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Area
Mean Pixel Value
Rawintden of the Drop
Rawintden of the Background
Trial 1
0
91382
71.613
6544125
18757
0.25
83776
40.875
3424354
2865
0.5
64632
64.815
4189144
373
1
72068
74.307
5355152
2461
2
74956
116.601
8739909
8280
5
81146
164.265
13329423
5734
Trial 2
0
84384
53.044
4476036
2665
0.25
60532
57.136
3458560
1701
0.5
88468
42.136
3727677
209
1
84626
85.175
7208044
106
2
87906
99.08
8709740
174
5
89884
162.199
14579066
87
Trial 3
0
97260
45.051
4381686
7168
0.25
92784
54.433
5050546
593
0.5
111748
73.74
8240277
783
1
88652
69.382
6150810
176
2
85960
100.872
8670951
175
5
93351
168.424
15722556
194
Fitting a Straight Line
Intends values for each Calf THymus DNA cocnentration.
PCR Results Summary
Instructor's summary: You completed 8 PCR reactions in a previous lab. You used the SYBR Green I staining and imaging technique to measure the amount of amplified DNA in each PCR reaction. You used a standard curve (based on known concnetration of claf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples are used to determine the threshold values for determining whether an unknown (patient) sample is truly positive or negative.
Your positive control PCR result was ____ μg/mL
Your negative control PCR result was ____ μg/mL
Write-in each patient ID and give both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed
Patient _____ :
Patient _____ :
Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion.
Patient _____ :
Patient _____ :