SYBR Green Dye [Instructions: A short summary describing SYBR green dye]
Single-Drop Fluorimeter [Instructions: A description of the single-drop fluorimeter device. Add a PHOTO for bonus points]
How the Fluorescence Technique Works [Instructions: In your own words]
Procedure
Smart Phone Camera Settings
Type of Smartphone: Apple iPhone 5S
Flash: Off
ISO setting: Auto
White Balance: Auto
Exposure: High
Saturation: High
Contrast: Low
Calibration
The camera was set up by placing the iPhone 5S in the holding compartment of the lab equipment. It was measured to be 6 centimeters away from the origin of the light emission. The settings (stated above) were adjusted; the height of the fluorimeter was adjusted in order to achieve a perfect side-angle perspective of the "beach ball" shaped liquid drop.
Distance between the smart phone cradle and drop = 8 centimeters
Solutions Used for Calibration
Initial Concentration of 2X Calf Thymus DNA solution (μg/mL)
Volume of the 2X DNA solution (μL)
Volume of the SYBR GREEN I
Final DNA concentration in SYBR Green I solution (μg/mL)
5
80
80
2.5
2
80
80
1.0
1
80
80
0.5
0.5
80
80
0.25
0.25
80
80
0.125
0
80
80
0
Placing Samples onto the Fluorimeter
Before placing samples onto the fluorimeter, turn on the Blue LED light & have the camera on the smartphone ready.
Insert the glass sampling surface, with the smooth side facing down.
Position the glass sampling surface so that the light emits between the first two rows of the glass.
Place an 80 μL drop of SYBR Green I in the middle of the first two rows.
Swap out the pipette tip with a new tip.
Place an 80 μL drop of a specifically concentrated Calf Thymus DNA solution onto the top of the previously placed SYBR Green I drop. Every three 160 μL sample drops will vary in concentrated Calf Thymus DNA solution.
In order to place a new sample drop, remove the 160 μL solution, move the glass sampling surface to the next two available rows, and repeat the process of placing two new 80 μL drops onto each other (as stated before).
Data Analysis
Representative Images of Samples
Sample w/o DNA
Sample w/ DNA
Image J Values for All Samples
Final DNA Concentration in SYBR Green I Solution (μg/mL)
Area (pixels)
Mean Pixel Value
RAWINTDEN of the Drop
RAWINTDEN of the Background
RAWINTDEN Drop - RAWINTDEN Background
0.5
60452
85.151
4421613
187151
4234462
0.5
59871
84.124
4521561
175412
4346149
0.5
60241
82.153
4159878
179854
3980024
1.0
63157
120.451
5904302
195454
5708848
1.0
64120
124.021
6048248
187945
5860303
1.0
63785
117.879
6248451
198454
6049997
2.5
73482
164.546
8558810
212416
8346394
2.5
74125
170.215
8821541
205451
8616090
2.5
73956
177.154
8258495
215411
8043084
0.0
56235
7.546
3201562
167842
3033720
0.0
56345
5.241
3158472
154952
3003520
0.0
57120
6.542
3584956
151231
3433725
0.125
57325
22.145
1124652
165451
959201
0.125
57694
24.564
1413586
167851
1245735
0.125
57120
27.156
1248412
170245
1078167
0.25
58503
78.151
3952152
168672
3783480
0.25
59871
84.4561
3754546
171541
3583005
0.25
59212
80.541
4132142
172164
3959978
Fitting a Straight Line
PCR Results Summary
PCR Product Tuble Label
Average INTDENS Value
PCR Product Concentration (μg/mL)
Corrected PCR Product Concentration (μg/mL)
A00
15659549.36
10.53189465
0.877657887
A01
6261410.862
2.952750695
0.246062558
A02
6193412.146
2.897913021
0.241492752
A03
7014809.498
3.56033024
0.296694187
B00
1885211.816
-0.576442084
-0.04803684
B01
8032454.681
4.38101184
0.36508432
B02
9363128.21
5.454135653
0.454511304
B03
10820943.92
6.629793484
0.55248279
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 concentrations of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples should be used as threshold values for determining whether an unknown (patient) sample is truly positive or negative.
Your positive control PCR result was 0.877657887 μg/mL
Your negative control PCR result was -0.04803684 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 _____ :